TW201925226A - Anti-CXCR5 antibodies and compositions and uses thereof - Google Patents

Abstract

The invention provides antibodies, and antigen-binding fragments thereof, that specifically bind to CXCR5. The antibodies can be afucosylated and exhibit increased ADCC compared with the otherwise identical fucosylated antibodies. The invention includes uses, and associated methods of using the antibodies.

Description

Anti-CXCR5 antibody and composition and uses thereof

The present invention relates to antibodies and antigen-binding fragments thereof that specifically bind to C-X-C chemokine receptor type 5 (CXCR5), as well as compositions, methods, and uses thereof. These antibodies are fucosylated and / or defucosylated and can exhibit varying effect functions.

CXC Chemokine Receptor Type 5 (CXCR5), also known as CD185 or Burkitt Lymphoma Receptor 1 (BLR1), is a naturally-occurring G protein-coupled receptor composed of B cells and bona fide ) Follicular T helper (Tfh) cells and circulating Tfh-like cells (herein interchangeably referred to as "cTfh" and "Tfh-like" cells). CXCR5 plays an important role in the immune response and is a potential target for the treatment of autoimmune diseases such as systemic lupus erythematosus (SLE).

As a key component of the humoral immune response, germinal center (GC) is a site where antigen-activated B cells proliferate, differentiate and undergo somatic cell hypermutation and the immunoglobulin (Ig) class switching of the antibodies they produce. Bona fide Tfh cells provide guidance signals to assist this process, which culminates in the production of affinity matured antibodies. The humoral "memory" of the GC reaction is maintained by long-term surviving plasma cells that maintain antibody concentration and memory B cells, which are triggered by the homology from memory Tfh cells after restimulation with antigens The secondary GC reaction resulted in more high-affinity plasma cells (McHeyzer-Williams et al., 2011, Nature Rev. Immunol. 12 (1): 24-34). Tfh circulating think like cells (hereinafter referred to as "Tfh-like" or "cTfh") after the re-encounter antigen also differentiate into real (bona fide) Tfh cells to support these memory responses (Crotty et al., 2011, Annu. Rev . Immmunol. 29: 621-663).

Importantly, the production of autoreactive B cells can also be caused by GC reactions, with undesirable consequences. In fact, a large number of chronic systemic autoimmune diseases such as SLE, RA, myositis, Sjögren's syndrome, ANCA-associated vasculitis, and scleroderma show signs of autoreactive humoral reactions. For example, many autoantibodies that are hallmarks of these diseases have high affinity, somatic mutation and Ig conversion, indicating that they are caused by autoreactive GC reactions (Vinuesa et al., 2009, Nature Rev. Immunol. 9 ( 12): 845-857). In addition, the frequency of circulating Tfh-like cells has been detected in the peripheral blood of patients with many of these autoimmune diseases, and their levels are often related to autoantibody titers and / or disease severity (Tangye et al., 2013, Nature Rev. Immunol. 13 (6): 412-426). Taken together, these data highlight B cells, bona fide Tfh cells, and circulating Tfh-like cells as possible therapeutic targets for many systemic autoimmune diseases.

CXCR5 is expressed by B cells, bona fide Tfh cells and circulating Tfh-like cells and mediates its migration along the gradient of CXCR5 ligand CXCL13 to GC reaction and participates in GC reaction (Vinuesa and Cyster, 2011, Immunity 35 (5): 671-680; Ansel et al., 1999, J. Exp. Med. 190 (8): 1123-1134; Ansel et al., 2000, Nature 406 (6793): 309-314; Cyster et al., 1999 , Curr. Top. Microbiol. Immunol. 246: 87-92; Hardtke et al., 2005, Blood 106 (6): 1924-1931; Haynes et al., 2007, J. Immunol. 179 (8): 5099-5108) . Therefore, targeting CXCR5 may have therapeutic benefits in treating autoimmune diseases.

In addition, targeting CXCR5 may also have therapeutic benefits in cancers characterized by the proliferation of cells expressing CXCR5, such as pancreatic cancer, colon cancer, bladder cancer, T-cell leukemia, and B-cell leukemia.

The present application discloses isolated antibodies and antigen-binding fragments that specifically bind to CXCR5 (C-X-C chemokine receptor type 5). In certain aspects, antibodies and antigen-binding fragments bind CXCR5 and reduce CXCR5 binding to CXCL13. In other aspects, the antibody may be fucosylated, but more preferably, it is defucosylated. In some aspects, antibodies and antigen-binding fragments exhibit varying effector functions. In certain aspects, antibodies and antigen-binding fragments thereof are defucosylated and exhibit increased antibody-dependent cytotoxicity compared to otherwise identical but fucosylated antibodies and antigen-binding fragments thereof ( ADCC).

In certain aspects, the present invention provides an isolated antibody and antigen-binding fragment thereof that specifically binds to CXCR5, wherein the antibody and antigen-binding fragment thereof contain the numbering according to SEQ ID NO: 32 at amino acid residue number 11 The epitope binding of Leucine (Leu; L) (L11).

In another aspect, the present invention provides an isolated antibody and antigen-binding fragment thereof that specifically binds to CXCR5, wherein the antibody and antigen-binding fragment thereof comprise the amino acid residue number 22 according to the numbering according to SEQ ID NO: 32 Epitope binding of aspartic acid (Asp; D).

Those skilled in the art will recognize or be able to determine many equivalents of specific embodiments of the invention described herein using only routine experimentation. Such equivalents are intended to be covered by the following embodiment (E).
E1. An isolated antibody or antigen-binding fragment thereof that specifically binds to CXC-chemokine receptor 5 (CXCR5), wherein the antibody or antigen-binding fragment thereof binds to human CXCR5 (hCXCR5) or cynomolgus monkey CXCR5 (cynoCXCR5) N Epitope in the domain.
E2. Such as the antibody or antigen-binding fragment of E1, wherein the antibody or antigen-binding fragment thereof binds to the epitope within amino acid residues 1-50 of hCXCR5 according to the amino acid sequence SEQ ID NO: 32, or to The epitope within amino acid residues 1-50 of cynoCXCR5 of SEQ ID NO: 33 is bound.
E3. The antibody or antigen-binding fragment thereof according to any one of E1 to E2, wherein the antibody or antigen-binding fragment thereof comprises leukamine at the amino acid residue number 11 according to the amino acid sequence SEQ ID NO: 32 Acid epitope.
E4. The antibody or antigen-binding fragment thereof according to any one of E1 to E3, wherein the antibody or antigen-binding fragment thereof binds asparagus at the amino acid residue number 22 according to the amino acid sequence SEQ ID NO: 32 The amino acid epitope.
E5. The antibody or antigen-binding fragment thereof according to any one of E1 to E4, wherein the antibody or antigen-binding fragment thereof comprises leukamine at the amino acid residue number 11 according to the amino acid sequence SEQ ID NO: 32 The epitope of the acid and aspartic acid at amino acid residue number 22.
E6. The antibody or antigen-binding fragment thereof according to any one of E1 to E5, wherein the antibody or antigen-binding fragment thereof comprises leukamine at the amino acid residue number 11 according to the amino acid sequence SEQ ID NO: 32 Acid, but does not bind hCXCR5 when the residue is not leucine.
E7. The antibody or antigen-binding fragment thereof according to any one of E1 to E6, wherein the antibody or antigen-binding fragment thereof comprises leukamine at the amino acid residue number 11 according to the amino acid sequence SEQ ID NO: 32 Acid, but does not bind hCXCR5 when the residue is threonine.
E8. The antibody or antigen-binding fragment thereof according to any one of E1 to E7, wherein the antibody or antigen-binding fragment thereof binds asparagus at the amino acid residue number 22 according to the amino acid sequence SEQ ID NO: 32 Amino acid, but does not bind hCXCR5 when the residue is not aspartic acid.
E9. The antibody or antigen-binding fragment thereof according to any one of E1 to E8, wherein the antibody or antigen-binding fragment thereof binds asparagus at the amino acid residue number 22 according to the amino acid sequence SEQ ID NO: 32 Amino acid, but does not bind hCXCR5 when the residue is alanine.
E10. The antibody or antigen-binding fragment thereof according to any one of E1 to E9, wherein the antibody or antigen-binding fragment thereof comprises leukamine at the amino acid residue number 11 according to the amino acid sequence SEQ ID NO: 32 Acid and aspartic acid at amino acid residue number 22, but does not bind the epitope in which the leucine is substituted with threonine and / or the aspartic acid is substituted with alanine.
E11. An antibody or antigen-binding fragment thereof according to any one of E1 to E10, wherein the antibody or antigen-binding fragment thereof exhibits an apparent affinity binding to human B cells with an EC50 of about 6.60 pM and a standard deviation of about ± 2.33 pM hCXCR5.
E12. The antibody or antigen-binding fragment thereof according to any one of E1 to E11, wherein the antibody or antigen-binding fragment thereof binds to human circulating follicular T with an apparent affinity of EC50 of about 5.89 pM and a standard deviation of about ± 1.40 pM HCXCR5 expressed on helper-like (Tfh-like) cells.
E13. The antibody or antigen-binding fragment thereof according to any one of E1 to E12, wherein the antibody or antigen-binding fragment binds to human follicular T helper-like (Tfh-like) cells with an apparent affinity of EC50 of about 10.6 pM HCXCR5.
E14. The antibody or antigen-binding fragment thereof according to any one of E1 to E13, wherein the antibody or antigen-binding fragment thereof binds cynoCXCR5 expressed on cynomolgus monkey B cells with an apparent affinity of EC50 of about 1.32 pM.
E15. The antibody or antigen-binding fragment thereof according to any one of E1 to E14, wherein the antibody or antigen-binding fragment thereof binds cynoCXCR5 expressed on cynomolgus monkey Tfh-like cells with an apparent affinity of EC50 of about 10.5 pM.
E16. The antibody or antigen-binding fragment of any one of E1 to E15, wherein the antibody or antigen-binding fragment thereof antagonizes CXCR5-CXCL13 signaling in cAMP report analysis with an EC50 of about 961 pM.
E17. The antibody or antigen-binding fragment thereof of any one of E1 to E16, wherein the antibody or antigen-binding fragment thereof exhibits ADCC activity against human B cells exhibiting an EC50 of about 2.01 pM with a standard deviation of about ± 2.28 pM.
E18. The antibody or antigen-binding fragment thereof according to any one of E1 to E17, wherein the antibody or antigen-binding fragment thereof exhibits ADCC activity on human Tfh-like cells expressing hCXCR5 with an EC50 of about 4.28 pM and a standard deviation of about ± 2.88 pM .
E19. The antibody or antigen-binding fragment thereof according to any one of E1 to E18, wherein the antibody or antigen-binding fragment thereof exhibits ADCC activity of a human Tfh cell expressing hCXCR5 with an EC50 of about 0.11 pM.
E20. The antibody or antigen-binding fragment thereof according to any one of E1 to E19, wherein the antibody or antigen-binding fragment thereof exhibits an ADCC of about 15.3 pM with a standard deviation of about ± 11.7 pM for cynoCXCR5 expressing cynomolgus monkey B cells active.
E21. An antibody or antigen-binding fragment thereof according to any one of E1 to E20, wherein the antibody or antigen-binding fragment thereof binds hCXCR5 but does not detectably bind to human chemokine receptors CCR1, CCR2, CCR3, CCR4, CCR5 , CCR6, CCR7, CCR8, CCR9, CCR10, CMKLR1, CXCR3R1, CXCR1, CXCR2, CXCR3, CXCR4, CXCR6, CXCR7 and XCR1.
E22. The antibody or antigen-binding fragment thereof according to any one of E1 to E21, wherein the antibody or antigen-binding fragment depletes B cells in peripheral blood.
E23. For example, the antibody or antigen-binding fragment of E22, in which the depletion of B cells in peripheral blood is reversible.
E24. The antibody or antigen-binding fragment thereof according to any one of E1 to E23, wherein the antibody or antigen-binding fragment depletes Tfh-like cells in peripheral blood.
E25. The antibody or antigen-binding fragment of any one of E1 to E24, wherein the antibody or antigen-binding fragment depletes Tfh cells in the spleen.
E26. The antibody or antigen-binding fragment thereof according to any one of E1 to E24, wherein the antibody or antigen-binding fragment thereof impairs the humoral immune memory response.
E27. For example, the antibody or antigen-binding fragment of E26, in which the damaged humoral immune memory response is against tetanus toxoid in cynomolgus monkeys.
E28. The antibody or antigen-binding fragment thereof according to any one of E1 to E27, wherein the antibody or antigen-binding fragment thereof inhibits CXCR5 from binding to CXCL13.
E29. An antibody or antigen-binding fragment thereof according to any one of E1 to E28, wherein the antibody or antigen-binding fragment thereof inhibits CXCL13 inhibition of cAMP production in cells that are otherwise triggered by forskolin, thereby Compared with the level of cAMP in the presence of antigen-binding fragments, the level of cAMP is increased.
E30. An antibody or antigen-binding fragment thereof according to any one of E1 to E29, wherein the antibody or antigen-binding fragment thereof inhibits CXCL13 production by cAMP with an EC50 of about 961 pM.
E31. For example, the antibody or antigen-binding fragment of E30, wherein the maximum inhibition of CXCL13 inhibition is at least about 60%, 70%, or 80%.
E32. An antibody or antigen-binding fragment thereof according to any one of E1 to E31, wherein the antibody or antigen-binding fragment thereof binds human B cells expressing CXCR5 (eg, CXCR5 + human B cells) with an apparent affinity of EC50 less than about 26 pM, but Does not bind to cells expressing CXCR5 mouse, rat, or rabbit heterologs.
E33. The antibody or antigen-binding fragment thereof according to any one of E1 to E32, wherein the antibody or antigen-binding fragment triggers human donor and cynomolgus monkey peripheral blood mononuclear cells (PBMC) and human donor tonsil monocytes (TMC ) In CXCR5 express ADCC of cells.
E34. An isolated antibody that specifically binds hCXCR5 and competes with the antibody or antigen-binding fragment of any one of E1-E33.
E35. An antibody or antigen-binding fragment thereof according to any one of E1 to E34, wherein the antibody or antigen-binding fragment thereof binds CXCR5 + human B cells with an apparent affinity of EC50 less than about 26 pM, but does not substantially bind to express CXCR5 Source cells. In some embodiments, the antibody or antigen-binding fragment thereof, for example, based on any of the analyses described herein, exhibits undetectable binding to cells expressing CXCR5 xenologs; or the EC50 is at least greater than the EC50 binding to hCXCR5 The 10,000-fold apparent affinity binds to cells expressing CXCR5 xenologs.
E36. The antibody or antigen-binding fragment thereof according to any one of E1 to E35, wherein the antibody or antigen-binding fragment thereof comprises: CDR-L1 comprising the amino acid sequence SEQ ID NO: 2; comprising the amino acid sequence SEQ ID NO: CDR-L2 of 3; CDR-L3 of amino acid sequence SEQ ID NO: 4; CDR-H1 of amino acid sequence SEQ ID NO: 7; CDR-H2 of amino acid sequence SEQ ID NO: 8 ; And CDR-H3 comprising the amino acid sequence SEQ ID NO: 9.
E37. The antibody or antigen-binding fragment thereof according to any one of E1 to E36, wherein the antibody or antigen-binding fragment thereof comprises: CDR-L1 comprising the amino acid sequence SEQ ID NO: 2; comprising the amino acid sequence SEQ ID NO: CDR-L2 of 3; CDR-L3 of amino acid sequence SEQ ID NO: 4; CDR-H1 of amino acid sequence SEQ ID NO: 7; CDR-H2 of amino acid sequence SEQ ID NO: 8 ; And CDR-H3 comprising the amino acid sequence SEQ ID NO: 11.
E38. The antibody or antigen-binding fragment thereof according to any one of E1 to E37, wherein the antibody or antigen-binding fragment thereof comprises: CDR-L1 comprising the amino acid sequence SEQ ID NO: 14; comprising the amino acid sequence SEQ ID NO: CDR-L2 of 15; CDR-L3 of amino acid sequence SEQ ID NO: 16; CDR-H1 of amino acid sequence SEQ ID NO: 19; CDR-H2 of amino acid sequence SEQ ID NO: 20 ; And CDR-H3 comprising the amino acid sequence SEQ ID NO: 21.
E39. The antibody or antigen-binding fragment thereof according to any one of E1 to E38, wherein the antibody or antigen-binding fragment thereof comprises: VL comprising an amino acid sequence selected from the group consisting of: amino acid sequence SEQ ID NO: 1 , SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 48-51, SEQ ID NO: 39 and SEQ ID NO: 58-62; VH free from the amino acid sequence of the group consisting of: amino acid sequence SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO : 36, SEQ ID NO: 40, SEQ ID NO: 53-57 and SEQ ID NO: 63.
E40. The antibody or antigen-binding fragment thereof according to any one of E1 to E39, wherein the antibody or antigen-binding fragment thereof comprises: VL comprising an amino acid sequence selected from the group consisting of: amino acid sequence SEQ ID NO: 1 , SEQ ID NO: 5, SEQ ID NO: 13, SEQ ID NO: 35, SEQ ID NO: 37, SEQ ID NO: 48-51, SEQ ID NO: 39 and SEQ ID NO: 58-62; VH free from the amino acid sequence of the group consisting of: amino acid sequence SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO : 36, SEQ ID NO: 40, SEQ ID NO: 53-57 and SEQ ID NO: 63.
E41. The antibody or antigen-binding fragment thereof according to any one of E1 to E40, wherein the antibody or antigen-binding fragment thereof comprises: an amino acid sequence encoded by an insert of a plastid with the deposit number PTA-124324 deposited with the ATCC VL and VH containing the amino acid sequence encoded by the insert deposited with ATCC and having the plastid with registration number PTA-124323.
E42. The antibody or antigen-binding fragment thereof according to any one of E1 to E35 and E39 to E40, wherein the antibody or antigen-binding fragment thereof comprises: a VL comprising the amino acid sequence SEQ ID NO: 13, and an amino acid sequence SEQ ID NO: 17 of VH.
E43. The antibody or antigen-binding fragment thereof according to any one of E1 to E35 and E38 to E40, wherein the antibody or antigen-binding fragment thereof comprises: a VL comprising the amino acid sequence SEQ ID NO: 13, and an amino acid sequence SEQ ID NO: 18 of VH.
E44. The antibody or antigen-binding fragment thereof according to any one of E1 to E35 and E38 to E40, wherein the antibody or antigen-binding fragment thereof comprises: VL comprising the amino acid sequence SEQ ID NO: 37, and comprising the amino acid sequence SEQ ID NO: 38 of VH.
E45. The antibody or antigen-binding fragment thereof according to any one of E1 to E36 and E39 to E41, wherein the antibody or antigen-binding fragment thereof comprises: a VL comprising the amino acid sequence SEQ ID NO: 35, and an amino acid sequence SEQ ID NO: 36 of VH.
E46. The antibody or antigen-binding fragment thereof according to any one of E1 to E36 and E39 to E41, wherein the antibody or antigen-binding fragment thereof comprises: VL comprising the amino acid sequence SEQ ID NO: 47, and comprising the amino acid sequence SEQ ID NO: 52 of VH.
E47. The antibody or antigen-binding fragment thereof according to any one of E1 to E36 and E39 to E41, wherein the antibody or antigen-binding fragment thereof comprises: a VL comprising the amino acid sequence SEQ ID NO: 5, and an amino acid sequence SEQ ID NO: 6 of VH.
E48. The antibody or antigen-binding fragment thereof according to any one of E1 to E36 and E39 to E41, wherein the antibody or antigen-binding fragment thereof comprises: a VL comprising the amino acid sequence SEQ ID NO: 5, and an amino acid sequence SEQ ID NO: 10 of VH.
E49. The antibody or antigen-binding fragment thereof according to any one of E1 to E36 and E39 to E41, wherein the antibody or antigen-binding fragment thereof comprises: a VL comprising the amino acid sequence SEQ ID NO: 1, and an amino acid sequence SEQ ID NO: 6 of VH.
E50. The antibody or antigen-binding fragment thereof according to any one of E1 to E35, E37, and E39 to E41, wherein the antibody or antigen-binding fragment thereof comprises: a VL comprising the amino acid sequence SEQ ID NO: 1, and an amino acid VH of sequence SEQ ID NO: 12.
E51. The antibody or antigen-binding fragment thereof according to any one of E1 to E35, E37, and E39 to E41, wherein the antibody or antigen-binding fragment thereof comprises: a VL comprising the amino acid sequence SEQ ID NO: 39, and an amino acid VH of sequence SEQ ID NO: 40.
E52. The antibody or antigen-binding fragment thereof according to any one of E1 to E35, E37, and E39 to E41, wherein the antibody or antigen-binding fragment thereof comprises: an LC comprising the amino acid sequence SEQ ID NO: 22, and an amino acid HC of sequence SEQ ID NO: 23.
E53. The antibody or antigen-binding fragment thereof according to any one of E1 to E35 and E38 to E40, wherein the antibody or antigen-binding fragment thereof comprises: an LC comprising the amino acid sequence SEQ ID NO: 24, and an amino acid sequence SEQ ID NO: 25 of HC.
E54. The antibody or antigen-binding fragment thereof according to any one of E1 to E35, E37, and E39 to E41, wherein the antibody or antigen-binding fragment thereof comprises: an LC comprising the amino acid sequence SEQ ID NO: 26, and an amino acid HC of sequence SEQ ID NO: 27.
E55. The antibody or antigen-binding fragment thereof according to any one of E1 to E35, E37 and E39 to E41, wherein the antibody or antigen-binding fragment thereof comprises: an LC comprising the amino acid sequence SEQ ID NO: 28, and an amino acid HC of sequence SEQ ID NO: 29.
E56. The antibody or antigen-binding fragment thereof according to any one of E1 to E35, E37, and E39 to E41, wherein the antibody or antigen-binding fragment thereof comprises VL encoded by the nucleic acid sequence SEQ ID NO: 95, and the nucleic acid sequence SEQ ID NO : 96 encoded VH.
E57. The antibody or antigen-binding fragment thereof according to any one of E1 to E35, E37, and E39 to E41, wherein the antibody or antigen-binding fragment thereof comprises LC encoded by the nucleic acid sequence SEQ ID NO: 97, and the nucleic acid sequence SEQ ID NO : 98 coded HC.
E58. The antibody or antigen-binding fragment thereof according to any one of E1 to E57, wherein the antibody or antigen-binding fragment thereof comprises a VL framework sequence and a VH framework sequence, and wherein one or both of the VL framework sequence or the VH framework sequence and its Human germline sequence in which the derived human germline sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, and in which the VL framework sequence is derived The VL sequence is selected from the group consisting of: DPK9, DPK12, DPK18, DPK24, HK102_V1, DPK1, DPK8, DPK3, DPK21, Vg_38K, DPK22, DPK15, DPL16, DPL8, V1-22, Vλ shared, Vλ1 shared, Vλ3 shared , Vκ consensus, Vκ1 consensus, Vκ2 consensus and Vκ3, and the human germline VH sequence from which the VH framework sequence is derived is selected from the group consisting of: DP54, DP47, DP50, DP31, DP46, DP71, DP75, DP10, DP7, DP49, DP51, DP38, DP79, DP78, DP73, VH3, VH5, VH1 and VH4.
E59. The antibody or antigen-binding fragment thereof according to any one of E1 to E58, wherein the antibody or antigen-binding fragment thereof is defucosylated.
E60. An antibody or antigen-binding fragment thereof such as E59, wherein the antibody or antigen-binding fragment thereof exhibits enhanced ADCC.
E61. An isolated antibody or antigen-binding fragment thereof, comprising CDR-H1, CDR-H2, and CDR-H3 sequences of a VH comprising an amino acid sequence selected from the group consisting of: amino acid sequence SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 25, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 23, SEQ ID NO: 27, SEQ ID NO: 52, SEQ ID NO : 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57 and SEQ ID NO: 63.
E62. An isolated antibody or antigen-binding fragment thereof comprising CDR-L1, CDR-L2 and CDR-L3 sequences of VL comprising an amino acid sequence selected from the group consisting of: amino acid sequence SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 13, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO : 50, SEQ ID NO: 51, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61 and SEQ ID NO: 62.
E63. The antibody or antigen-binding fragment thereof according to any one of E1 to E62, which comprises the CDR-H1, CDR-H2, and CDR-H3 sequences as set forth in the amino acid sequence SEQ ID NO: 6.
E64. The antibody or antigen-binding fragment thereof according to any one of E1 to E62, which includes the CDR-L1, CDR-L2, and CDR-L3 sequences of the amino acid sequence SEQ ID NO: 1.
E65. An antibody or antigen-binding fragment thereof according to any one of E1 to E64, which contains one or more of the following amino acid substitutions:
1, 2, 3, 4, 5 or 6 substitutions in CDR-L1 for the corresponding residues of the human germline VL sequence,
1, 2, 3, 4 or 5 substitutions in CDR-L2 for the corresponding residues of the human VL germline sequence,
1, 2, 3, 4, 5 or 6 substitutions in CDR L3 for the corresponding residues of the human germline VL sequence,
1, 2, 3, 4, 5 or 6 substitutions in CDR-H1 for the corresponding residues of the human germline VH sequence,
1, 2, 3, 4, 5, 6, 7 or 8 substitutions in CDR H2 for the corresponding residues of the human germline VH sequence,
The human reproductive VL sequence is selected from the group consisting of: DPK9, DPK12, DPK18, DPK24, HK102_V1, DPK1, DPK8, DPK3, DPK21, Vg_38K, DPK22, DPK15, DPL16, DPL8, V1-22, Vκ1 common, Vκ2 Shared with Vκ3, and the human reproductive system VH is selected from the group consisting of DP54, DP47, DP50, DP31, DP46, DP71, DP75, DP10, DP7, DP49, DP51, DP38, DP79, DP78, DP73, VH3, VH5, VH1 and VH4.
E66. The antibody or antigen-binding fragment thereof according to any one of E61 to E65, which comprises a VH framework sequence derived from a human germline VH sequence selected from the group consisting of: DP54, DP47, DP50, DP31 , DP46, DP71, DP75, DP10, DP7, DP49, DP51, DP38, DP79, DP78, DP73, VH3, VH5, VH1 and VH4.
E67. The antibody or antigen-binding fragment of any one of E61 to E66, which comprises a framework VH sequence derived from human VH3 germline sequence.
E68. The antibody or antigen-binding fragment thereof of any one of E61 to E67, which comprises a framework VH sequence derived from a human germline VH sequence selected from the group consisting of DP54, DP47, DP50, DP31, DP46, DP49, and DP51.
E69. The antibody or antigen-binding fragment thereof of any one of E61 to E68, which comprises a framework VH sequence derived from a human germline VH sequence selected from the group consisting of DP54, DP47, DP50, and DP31.
E70. The antibody or antigen-binding fragment thereof according to any one of E61 to E69, which comprises a VH framework sequence derived from a human reproductive line DP54 sequence.
E71. An antibody or antigen-binding fragment thereof according to any one of E61 to E70, which comprises a VL framework sequence derived from a human germline VL sequence selected from the group consisting of: DPK9, DPK12, DPK18, DPK24, HK102_V1, DPK1, DPK8, DPK3, DPK21, Vg_38K, DPK22, DPK15, DPL16, DPL8, V1-22, Vκ shared, Vκ1 shared, Vκ2 shared, and Vκ3 shared.
E72. The antibody or antigen-binding fragment thereof according to any one of E61 to E71, which comprises a VL framework sequence derived from a human germline VL sequence selected from the group consisting of: DPK9, DPK12, DPK18, DPK24, HK102_V1, DPK1, DPK8, DPK3, DPK21, Vg_38K, DPK22, DPK15, Vκ shared, Vκ1 shared, Vκ2 shared, and Vκ3.
E73. The antibody or antigen-binding fragment thereof according to any one of E61 to E72, which includes a VL framework sequence derived from a human reproductive system Vκ1 sequence.
E74. The antibody or antigen-binding fragment of any one of E61 to E73, which comprises a VL framework sequence derived from a human germline VL sequence selected from the group consisting of: DPK9, HK102_V1, DPK1, and DPK8.
E75. The antibody or antigen-binding fragment thereof according to any one of E61 to E74, which comprises a VL framework sequence derived from a human reproductive system DPK9 sequence.
E76. The antibody or antigen-binding fragment thereof according to any one of E61 to E75, which comprises a VL framework sequence and a VH framework sequence, and wherein one or both of the VL framework sequence or the VH framework sequence and the human germline sequence derived therefrom At least 90% consistent.
E77. The antibody or antigen-binding fragment thereof according to any one of E61 to E76, which comprises a VL framework sequence and a VH framework sequence, and wherein one or both of the VL framework sequence or VH framework sequence and the human germline sequence derived therefrom At least 66%, 76%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% are consistent.
E78. The antibody or antigen-binding fragment thereof according to any one of E61 to E77, which comprises a VL framework sequence and a VH framework sequence, and wherein one or both of the VL framework sequence or VH framework sequence and the human germline sequence derived therefrom Consistent.
E79. The antibody or antigen-binding fragment of any one of E61 to E78, which comprises VH, the VH comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 6.
E80. The antibody or antigen-binding fragment thereof of any one of E61 to E79, which comprises a VH, the VH comprising an amino acid sequence that is at least 92% identical to SEQ ID NO: 6.
E81. The antibody or antigen-binding fragment thereof according to any one of E61 to E80, which comprises VH, and the VH comprises the amino acid sequence SEQ ID NO: 6.
E82. The antibody or antigen-binding fragment thereof according to any one of E61 to E81, which comprises a VL comprising an amino acid sequence that is at least 66% identical to SEQ ID NO: 1.
E83. The antibody or antigen-binding fragment thereof according to any one of E61 to E82, which comprises VL, which comprises at least 66%, 76%, 80%, 91%, 92%, 93%, 94% of SEQ ID NO: 1 , 95%, 96%, 97%, 98% or 99% identical amino acid sequence
E84. The antibody or antigen-binding fragment thereof according to any one of E61 to E83, which comprises VL, and the VL comprises the amino acid sequence SEQ ID NO: 1.
E85. The antibody or antigen-binding fragment thereof according to any one of E1 to E84, which includes an Fc domain.
E86. An antibody such as E85 or an antigen-binding fragment thereof, wherein the Fc domain is an Fc domain of IgA (eg IgA1 or IgA2), IgD, IgE, IgM or IgG (eg IgG1, IgG2, IgG3 or IgG4).
E87. For example, the antibody or antigen-binding fragment of E86, wherein the Fc domain is the Fc domain of IgG.
E88. For example, the antibody or antigen-binding fragment of E87, wherein IgG is selected from the group consisting of IgG1, IgG2, IgG3 or IgG4.
E89. Such as E88 antibodies or antigen-binding fragments, of which IgG is IgG1.
E90. The antibody or antigen-binding fragment thereof according to any one of E1 to E89, which comprises a heavy chain comprising an amino acid sequence at least 90% identical to SEQ ID NO: 29.
E91. The antibody or antigen-binding fragment thereof according to any one of E1 to E89, which comprises a heavy chain comprising at least 91%, 92%, 93%, 94%, 95%, 96%, SEQ ID NO: 29, 97%, 98% or 99% identical amino acid sequence.
E92. The antibody or antigen-binding fragment thereof according to any one of E1 to E89, which includes a heavy chain including the amino acid sequence SEQ ID NO: 29.
E93. The antibody or antigen-binding fragment thereof of any one of E1 to E93, which comprises an LC comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 28.
E94. The antibody or antigen-binding fragment thereof according to any one of E1 to E93, which comprises an LC comprising at least 91%, 92%, 93%, 94%, 95%, 96%, 97% of SEQ ID NO: 28 , 98% or 99% identical amino acid sequence.
E95. The antibody or antigen-binding fragment thereof according to any one of E1 to E94, which includes an LC, and the LC includes an amino acid sequence SEQ ID NO: 28.
E96. An isolated antibody or antigen-binding fragment thereof, comprising a VH sequence encoded by an insert deposited with ATCC and having a plastid with ATCC accession number PTA-124323.
E97. An isolated antibody or antigen-binding fragment thereof comprising a VL sequence encoded by an insert deposited with ATCC and having a plastid with ATCC accession number PTA-124324.
E98. An antibody or antigen-binding fragment that competes with one or more of the following for binding to human CXCR5: mouse 11G2, chimeric 11G2, h11G2 VH (XC152) / VL (XC151), h11G2 VH (XC152) / VL ( XC153), h11G2 VH (XC152) / VL (XC154), h11G2 VH (XC152) / VL (XC346), h11G2 VH (XC152) / VL (XC347), h11G2 VH (XC152) / VL (XC348), h11G2 VH ( XC152) / VL (XC349), h11G2 VH (XC155) / VL (XC151), h11G2 VH (XC155) / VL (XC153), h11G2 VH (XC155) / VL (XC154), h11G2 VH (XC155) / VL (XC346) ), H11G2 VH (XC155) / VL (XC347), h11G2 VH (XC155) / VL (XC3484), h11G2 VH (XC155) / VL (XC349), h11G2 VH (XC156) / VL (XC151), h11G2 VH (XC156) ) / VL (XC153), h11G2 VH (XC156) / VL (XC154), h11G2 VH (XC156) / VL (XC346), h11G2 VH (XC156) / VL (XC347), h11G2 VH (XC156) / VL (XC348) , H11G2 VH (XC156) / VL (XC349), h11G2 VH (XC157) / VL (XC151), h11G2 VH (XC157) / VL (XC153), h11G2 VH (XC157) / VL (XC154), h11G2 VH (XC157) / VL (XC346), h11G2 VH (XC157) / VL (XC347), h11G2 VH (XC157) / VL (XC348), h11G2 VH (XC157) / VL (XC349), h11G2 VH (XC350) / VL (XC151), h11G2 VH (XC350) / VL (XC153), h11G2 VH (XC350) / VL (XC154), h11G2 VH (XC350) / VL (XC346), h11G2 VH (XC350) / VL (XC347), h11G2 VH (XC350) / VL (XC348), h11G2 VH (XC350) / VL (XC349), h11G2 VH (XC351) / VL (XC151), h11G2 VH (XC351) / VL (XC153), h11G2 VH (XC351) / VL (XC154), h11G2 VH (XC351) / VL (XC346), h11G2 VH (XC351) / VL (XC347), h11G2 VH (XC351) / VL (XC348), and h11G2 VH (XC351) / VL (XC349), h11G2 VH (XC352) / VL (XC151), h11G2 VH (XC352) / VL (XC153), h11G2 VH (XC352) / VL (XC154), h11G2 VH (XC352) / VL (XC346), h11G2 VH (XC352) / VL (XC347), h11G2 VH (XC352) / VL (XC348), h11G2 VH (XC352) / VL (XC349), h11G2 VH (XC353) / VL (XC151), h11G2 VH (XC353) / VL (XC153), h11G2 VH (XC353) / VL (XC154), h11G2 VH (XC353) / VL (XC346), h11G2 VH (XC353) / VL (XC347), h11G2 VH (XC353) / VL (XC348), h11G2 VH (XC353) / VL (XC349), h11G2 VH (XC354) / VL (XC151), h11G2 VH (XC354) / VL (XC153), h11G2 VH (XC354) / VL (XC154), h11G2 VH (XC354) / VL (XC346), h11G2 VH (XC354) / VL ( (XC347), h11G2 VH (XC354) / VL (XC348), h11G2 VH (XC354) / VL (XC349), mouse 41A10, chimeric 41A10, h41A10 VH (XC147) / VL (XC142), h41A10 VH (XC147) / VL (XC143), h41A10 VH (XC147) / VL (XC144), h41A10 VH (XC147) / VL (XC145), h41A10 VH (XC147) / VL (XC146), h41A10 VH (XC147) / VL (XC149), h41A10 VH (XC148) / VL (XC142), h41A10 VH (XC148) / VL (XC143), h41A10 VH (XC148) / VL ( XC144), h41A10 VH (XC148) / VL (XC145), h41A10 VH (XC148) / VL (XC146), h41A10 VH (XC148) / VL (XC149), h41A10 VH (XC150) / VL (XC142), h41A10 VH ( XC150) / VL (XC143), h41A10 VH (XC150) / VL (XC144), h41A10 VH (XC150) / VL (XC145), h41A10 VH (XC150) / VL (XC146), h41A10 VH (XC150) / VL (XC149 ), Mouse 5H7, and chimeric 5H7.
E99. An antibody or antigen-binding fragment thereof that competes with the antibody of any one of E1-E98 for binding to human CXCR5 and CXCL13.
E100. The antibody or antigen-binding fragment thereof according to any one of E1 to E99, wherein the antibody or antigen-binding fragment is an Fc fusion protein, a monofunctional antibody, a maximum antibody, a bifunctional antibody, scFab, scFv, or a peptibody.
E101. An antibody or antigen-binding fragment thereof such as E1 to E100, wherein the antibody or antigen-binding fragment thereof binds to human CXCR5 at a value approximately equal to or less than that selected from the group consisting of approximately 10 nM, 5 nM, 2 nM, 1 nM, 900 pM, 800 pM, 700pM, 600pM, 500pM, 400pM, 300pM, 250pM, 200pM, 150pM, 100pM, 50pM, 40pM, 30pM, 25pM, 20pM, 15pM, 10pM, 5pM and 1pM.
E102. An antibody or antigen-binding fragment thereof such as E1 to E101, wherein the antibody or antigen-binding fragment thereof binds to cynomolgus cynomolgus monkey CXCR5 at a value approximately equal to or less than that selected from the group consisting of: approximately 10 nM, 5 nM, 2 nM, 1 nM, 900 pM, 800pM, 700pM, 600pM, 500pM, 400pM, 300pM, 250pM, 200pM, 150pM, 100pM, 50pM, 40pM, 30pM, 25pM, 20pM, 15pM, 13pM, 10pM, 5pM and 1pM.
E103. For example, antibodies or antigen-binding fragments of E1 to E102, wherein the binding KD of the antibody or antigen-binding fragment to cynomolgus monkey CXCR5 is within one order of magnitude of the binding KD of the antibody or antigen-binding fragment to human CXCR5.
E104. For example, the antibody or antigen-binding fragment of E1 to E103, wherein the ratio of the antibody or antigen-binding fragment to the human CXCR5 binding KD compared to the rhesus monkey CXCR5 binding is between 5: 1 and 1: 5.
E105. For example, the antibody or antigen-binding fragment of E1 to E104, wherein the ratio of the antibody or antigen-binding fragment to human CXCR5 binding KD compared to the cynomolgus monkey CXCR5 binding is between 2: 1 and 1: 2.
E106. For example, the antibody or antigen-binding fragment of E1 to E105, wherein the ratio of the antibody or antigen-binding fragment to the rhesus monkey CXCR5 binding KD compared to the human CXCR5 binding is within the following range: the lower limit is selected from the group consisting of 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5 and 6.2, and the upper limit value is selected from the group consisting of: 1.4, 1.5 , 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0 , 4.1, 4.2, 4.3, 4.4, 4.5, 6.2, 9, 9.2 and 10.
E107. For example, the antibody or antigen-binding fragment of E1 to E106, wherein the ratio of the antibody or antigen-binding fragment to the KD of cynomolgus monkey CXCR5 of SEQ ID NO: 33 compared to the binding of human CXCR5 of SEQ ID NO: 32 is about 1.0 Between about 10.0.
E108. The antibody or antigen-binding fragment of any one of E1 to 107, wherein the predicted half-life in humans is in the range of about one (1) day to twenty-one (21) days.
E109. The antibody or antigen-binding fragment of any one of E1 to 108, wherein the predicted half-life in humans is about seventeen (17) days.
E110. An antibody or antigen-binding fragment thereof, which comprises a CDR selected from the group consisting of an antibody or antigen-binding fragment thereof: mouse 11G2 VH, mouse 11G2 VL, chimeric 11G2 VH, chimeric 11G2 VL, humanized 11G2 VH (XC152), h11G2 VH (XC155), h11G2 VH (XC156), h11G2 VH (XC157), h11G2 VH (XC350), h11G2 VH (XC351), h11G2 VH (XC352), h11G2 VH (XC353), h11G2 VH (C ), H11G2 VL (XC151), h11G2 VL (XC153), h11G2 VL (XC154), h11G2 VL (XC346), h11G2 VL (XC347), h11G2 VL (XC348), h11G2 VL (XC349), mouse 41A10 VH, small Mouse 41A10 VL, chimeric 41A10 VH, chimeric 41A10 VL, humanized 41A10 VH (XC147), h41A10 VH (XC148), h41A10 VH (XC150), h41A10 VL (XC142), h41A10 VL (XC143), h41A10 VL (XC144) ), H41A10 VL (XC145), h41A10 VL (XC146), h41A10 VL (XC149), mouse 5H7 VH, mouse 5H7 VL, chimeric 5H7 VH and chimeric 5H7 VL.
E111. An antibody or antigen-binding fragment thereof comprising VL and VH selected from the group consisting of antibody or antigen-binding fragment thereof: mouse 11G2 VH, mouse 11G2 VL, chimeric 11G2 VH, chimeric 11G2 VL, h11G2 VH (XC152), h11G2 VH (XC155), h11G2 VH (XC156), h11G2 VH (XC157), h11G2 VH (XC350), h11G2 VH (XC351), h11G2 VH (XC352), h11G2 VH (XC353), h11G2 VH (C ), H11G2 VL (XC151), h11G2 VL (XC153), h11G2 VL (XC154), h11G2 VL (XC346), h11G2 VL (XC347), h11G2 VL (XC348), h11G2 VL (XC349), mouse 41A10 VH, small Mouse 41A10 VL, chimeric 41A10 VH, chimeric 41A10 VL, humanized 41A10 VH (XC147), h41A10 VH (XC148), h41A10 VH (XC150), h41A10 VL (XC142), h41A10 VL (XC143), h41A10 VL (XC144) ), H41A10 VL (XC145), h41A10 VL (XC146), h41A10 VL (XC149), mouse 5H7 VH, mouse 5H7 VL, chimeric 5H7 VH, and chimeric 5H7 VL.
E112. An antibody or antigen-binding fragment thereof selected from the group consisting of:
a. Antibodies containing mouse 11G2 VH and mouse 11G2 VL;
b. Antibodies containing chimeric 11G2 VH and chimeric 11G2 VL;
c. Antibodies containing the following: humanized 11G2 VH (XC152) and VL selected from the group consisting of: mouse 11G2 VL, chimeric 11G2 VL, h11G2 VL (XC151), h11G2 VL (XC153), h11G2 VL (XC154), h11G2 VL (XC346), h11G2 VL (XC347), h11G2 VL (XC348) and h11G2 VL (XC349);
d. Antibodies containing the following: humanized 11G2 VH (XC155), and VL selected from the group consisting of mouse 11G2 VL, chimeric 11G2 VL, h11G2 VL (XC151), h11G2 VL (XC153), h11G2 VL (XC154), h11G2 VL (XC346), h11G2 VL (XC347), h11G2 VL (XC348) and h11G2 VL (XC349);
e. Antibodies containing the following: humanized 11G2 VH (XC156) and VL selected from the group consisting of mouse 11G2 VL, chimeric 11G2 VL, h11G2 VL (XC151), h11G2 VL (XC153), h11G2 VL (XC154), h11G2 VL (XC346), h11G2 VL (XC347), h11G2 VL (XC348) and h11G2 VL (XC349);
f. Antibodies containing the following: humanized 11G2 VH (XC157) and VL selected from the group consisting of: mouse 11G2 VL, chimeric 11G2 VL, h11G2 VL (XC151), h11G2 VL (XC153), h11G2 VL (XC154), h11G2 VL (XC346), h11G2 VL (XC347), h11G2 VL (XC348) and h11G2 VL (XC349);
g. Antibodies containing the following: humanized 11G2 VH (XC350) and VL selected from the group consisting of: mouse 11G2 VL, chimeric 11G2 VL, h11G2 VL (XC151), h11G2 VL (XC153), h11G2 VL (XC154), h11G2 VL (XC346), h11G2 VL (XC347), h11G2 VL (XC348) and h11G2 VL (XC349);
h. Antibodies containing the following: h11G2 VH (XC351) and VL selected from the group consisting of: mouse 11G2 VL, chimeric 11G2 VL, h11G2 VL (XC151), h11G2 VL (XC153), h11G2 VL (XC154) ), H11G2 VL (XC346), h11G2 VL (XC347), h11G2 VL (XC348) and h11G2 VL (XC349);
i. Antibodies containing: h11G2 VH (XC352) and VL selected from the group consisting of: mouse 11G2 VL, chimeric 11G2 VL, h11G2 VL (XC151), h11G2 VL (XC153), h11G2 VL (XC154) ), H11G2 VL (XC346), h11G2 VL (XC347), h11G2 VL (XC348) and h11G2 VL (XC349);
j. Antibodies containing the following: h11G2 VH (XC353) and VL selected from the group consisting of: mouse 11G2 VL, chimeric 11G2 VL, h11G2 VL (XC151), h11G2 VL (XC153), h11G2 VL (XC154) ), H11G2 VL (XC346), h11G2 VL (XC347), h11G2 VL (XC348) and h11G2 VL (XC349);
k. Antibodies containing the following: h11G2 VH (XC354) and VL selected from the group consisting of: mouse 11G2 VL, chimeric 11G2 VL, h11G2 VL (XC151), h11G2 VL (XC153), h11G2 VL (XC154) ), H11G2 VL (XC346), h11G2 VL (XC347), h11G2 VL (XC348) and h11G2 VL (XC349);
l. Antibodies containing the following: h11G2 VL (XC151), and VH selected from the group consisting of mouse 11G2 VH, chimeric 11G2 VH, h11G2 VH (XC152), h11G2 VH (XC155), h11G2 VH ( XC156), h11G2 VH (XC157), h11G2 VH (XC350), h11G2 VH (XC351), h11G2 VH (XC352), h11G2 VH (XC353) and h11G2 VH (XC354);
m. Antibodies including: h11G2 VL (XC153), and VH selected from the group consisting of mouse 11G2 VH, chimeric 11G2 VH, h11G2 VH (XC152), h11G2 VH (XC155), h11G2 VH ( XC156), h11G2 VH (XC157), h11G2 VH (XC350), h11G2 VH (XC351), h11G2 VH (XC352), h11G2 VH (XC353) and h11G2 VH (XC354);
n. Antibodies containing the following: h11G2 VL (XC154), and VH selected from the group consisting of mouse 11G2 VH, chimeric 11G2 VH, h11G2 VH (XC152), h11G2 VH (XC155), h11G2 VH ( XC156), h11G2 VH (XC157), h11G2 VH (XC350), h11G2 VH (XC351), h11G2 VH (XC352), h11G2 VH (XC353) and h11G2 VH (XC354);
o. Antibodies containing the following: h11G2 VL (XC346), and VH selected from the group consisting of: mouse 11G2 VH, chimeric 11G2 VH, h11G2 VH (XC152), h11G2 VH (XC155), h11G2 VH ( XC156), h11G2 VH (XC157), h11G2 VH (XC350), h11G2 VH (XC351), h11G2 VH (XC352), h11G2 VH (XC353) and h11G2 VH (XC354);
p. Antibodies containing: h11G2 VL (XC347), and VH selected from the group consisting of mouse 11G2 VH, chimeric 11G2 VH, h11G2 VH (XC152), h11G2 VH (XC155), h11G2 VH ( XC156), h11G2 VH (XC157), h11G2 VH (XC350), h11G2 VH (XC351), h11G2 VH (XC352), h11G2 VH (XC353) and h11G2 VH (XC354);
q. Antibodies containing the following: h11G2 VL (XC348), and VH selected from the group consisting of mouse 11G2 VH, chimeric 11G2 VH, h11G2 VH (XC152), h11G2 VH (XC155), h11G2 VH ( XC156), h11G2 VH (XC157), h11G2 VH (XC350), h11G2 VH (XC351), h11G2 VH (XC352), h11G2 VH (XC353) and h11G2 VH (XC354);
r. Antibodies containing the following: h11G2 VL (XC349), and VH selected from the group consisting of mouse 11G2 VH, chimeric 11G2 VH, h11G2 VH (XC152), h11G2 VH (XC155), h11G2 VH ( XC156), h11G2 VH (XC157), h11G2 VH (XC350), h11G2 VH (XC351), h11G2 VH (XC352), h11G2 VH (XC353) and h11G2 VH (XC354);
s. Antibodies comprising: mouse 41A10 VH, and VL selected from the group consisting of: mouse 41A10 VL, chimeric 41A10 VL, h41A10 VL (XC142), h41A10 VL (XC143), h41A10 VL (XC144 ), H41A10 VL (XC145), h41A10 VL (XC146) and h41A10 VL (XC149);
t. Antibodies containing the following: chimeric 41A10 VH, and VL selected from the group consisting of: mouse 41A10 VL, chimeric 41A10 VL, h41A10 VL (XC142), h41A10 VL (XC143), h41A10 VL (XC144 ), H41A10 VL (XC145), h41A10 VL (XC146) and h41A10 VL (XC149);
u. Antibodies containing the following: humanized 41A10 VH (XC147), and VL selected from the group consisting of mouse 41A10 VL, chimeric 41A10 VL, h41A10 VL (XC142), h41A10 VL (XC143), h41A10 VL (XC144), h41A10 VL (XC145), h41A10 VL (XC146) and h41A10 VL (XC149);
v. Antibodies containing the following: h41A10 VH (XC148), and VL selected from the group consisting of mouse 41A10 VL, chimeric 41A10 VL, h41A10 VL (XC142), h41A10 VL (XC143), h41A10 VL ( XC144), h41A10 VL (XC145), h41A10 VL (XC146) and h41A10 VL (XC149);
w. h41A10 VH (XC150), and VL selected from the group consisting of: mouse 41A10 VL, chimeric 41A10 VL, h41A10 VL (XC142), h41A10 VL (XC143), h41A10 VL (XC144), h41A10 VL (XC145) ), H41A10 VL (XC146), h41A10 VL (XC149) and h41A10 VL (XC142);
x. Antibodies containing: mouse 41A10 VL, and VH selected from the group consisting of: mouse 41A10 VH, chimeric 41A10 VH, h41A10 VH (XC147), h41A10 VL (XC148) and h41A10 VL (XC150 );
y. Antibodies containing the following: chimeric 41A10 VL, and VH selected from the group consisting of: mouse 41A10 VH, chimeric 41A10 VH, h41A10 VH (XC147), h41A10 VL (XC148) and h41A10 VL (XC150 );
z. Antibodies containing the following: h41A10 VL (XC143), and VH selected from the group consisting of: mouse 41A10 VH, chimeric 41A10 VH, h41A10 VH (XC147), h41A10 VL (XC148) and h41A10 VL ( XC150);
aa. Antibodies containing: h41A10 VL (XC144), and VH selected from the group consisting of: mouse 41A10 VH, chimeric 41A10 VH, h41A10 VH (XC147), h41A10 VL (XC148) and h41A10 VL (a XC150);
bb. Antibodies containing the following: h41A10 VL (XC145), and VH selected from the group consisting of mouse 41A10 VH, chimeric 41A10 VH, h41A10 VH (XC147), h41A10 VL (XC148) and h41A10 VL ( XC150);
cc. Antibodies containing: h41A10 VL (XC146), and VH selected from the group consisting of: mouse 41A10 VH, chimeric 41A10 VH, h41A10 VH (XC147), h41A10 VL (XC148), and h41A10 VL ( XC150);
dd. Antibodies containing: h41A10 VL (XC149), and VH selected from the group consisting of mouse 41A10 VH, chimeric 41A10 VH, h41A10 VH (XC147), h41A10 VL (XC148), and h41A10 VL ( XC150);
ee. Antibodies containing the following: mouse 5H7 VH, and VL selected from the group consisting of mouse 5H7 and chimeric 5H7 VL;
ff. Mouse 5H7 VL, and VH selected from the group consisting of mouse 5H7 VH and chimeric 5H7 VH;
gg. Antibodies containing h11G2 VH (XC152) and h11G2 VL (XC151);
hh. Antibodies containing h11G2 VH (XC155) and h11G2 VL (XC153);
ii. Antibodies containing h11G2 VH (XC155) and h11G2 VL (XC154);
jj. Antibodies containing h11G2 VH (XC156) and h11G2 VL (XC153);
kk. Antibodies containing h11G2 VH (XC157) and h11G2 (XC154);
ll. Antibodies containing mouse 11G2 VH and mouse 11G2 VL;
mm. Antibodies containing chimeric 11G2 HC and chimeric 11G2 LC;
nn. Antibodies containing h11G2 VH (XC152) and h11G2 VL (XC151);
oo. Antibodies containing h11G2 VH (XC155) and h11G2 VL (XC154);
pp. Antibodies containing h11G2 VH (XC156) and h11G2 VL (XC153);
qq. Antibodies containing h11G2 VH (XC157) and h11G2 VL (XC154);
rr. Antibodies containing mouse 41A10 VH and mouse 41A10 VL;
ss. Antibodies containing chimeric 41A10 HC and chimeric 41A10 LC;
tt. Antibodies containing h41A10 VH (XC147) and h41A10 VL (XC142);
uu. Antibodies containing h41A10 VH (XC147) and h41A10 VL (XC143);
vv. Antibodies containing h41A10 VH (XC147) and h41A10 VL (XC144);
ww. Antibodies containing h41A10 VH (XC147) and h41A10 VL (XC145);
xx. Antibodies containing h41A10 VH (XC148) and h41A10 VL (XC142);
yy. Antibodies containing h41A10 VH (XC148) and h41A10 VL (XC143);
zz. Antibodies containing h41A10 VH (XC148) and h41A10 VL (XC144);
aaa. Antibodies containing mouse 5H7 VH and mouse 5H7 VL; and
bbb. Antibodies containing chimeric 5H7 HC and chimeric 5H7 LC.
E113. An antibody or antigen-binding fragment thereof selected from the group consisting of:
(a) Antibodies comprising: VH comprising the amino acid sequence SEQ ID NO: 36 and VL comprising the amino acid sequence SEQ ID NO: 35;
(b) an antibody comprising: CDR-H1 comprising the sequence SEQ ID: 7; CDR-H2 comprising the amino acid sequence SEQ ID NO: 8; CDR-H3 comprising the amino acid sequence SEQ ID NO: 9 ; CDR-L1 comprising the amino acid sequence SEQ ID NO: 2; CDR-L2 comprising the amino acid sequence SEQ ID NO: 3; and CDR-L3 comprising the sequence SEQ ID NO: 4;
(c) an antibody comprising: CDR-H1 comprising the sequence SEQ ID: 7; CDR-H2 comprising the amino acid sequence SEQ ID NO: 8; CDR-H3 comprising the amino acid sequence SEQ ID NO: 11 ; CDR-L1 comprising the amino acid sequence SEQ ID NO: 2; CDR-L2 comprising the amino acid sequence SEQ ID NO: 3; and CDR-L3 comprising the sequence SEQ ID NO: 4;
(d) An antibody comprising: CDR-H1 comprising the sequence SEQ ID NO: 19; CDR-H2 comprising the amino acid sequence SEQ ID NO: 20; CDR- comprising the amino acid sequence SEQ ID NO: 21 H3; CDR-L1 comprising the amino acid sequence SEQ ID NO: 14; CDR-L2 comprising the amino acid sequence SEQ ID NO: 15; and CDR-L3 comprising the sequence SEQ ID NO: 16;
(e) Antibodies containing: CDR-H1, CDR-H2, CDR-H3 as described in the amino acid sequence SEQ ID NO: 6, and as described in the amino acid sequence SEQ ID NO: 1 CDR-L1, CDR-L2 and CDR-L3;
(f) Antibodies containing the following: CDR-H1, CDR-H2, CDR-H3 encoded by an insert deposited with ATCC with a plastid with accession number PTA-124323, and with an accession number deposited with ATCC CDR-L1, CDR-L2 and CDR-L3 encoded by the plastid insert of PTA-124324;
(g) Antibodies comprising: VH (XC152) comprising the amino acid sequence SEQ ID NO: 52 and VL comprising the amino acid sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO : 5, SEQ ID NO: 35, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 51;
(h) Antibody comprising: VH (XC155) comprising the amino acid sequence SEQ ID NO: 6 and VL comprising the amino acid sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO : 5, SEQ ID NO: 35, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 51;
(i) Antibodies comprising: VH (XC156) comprising the amino acid sequence SEQ ID NO: 10 and VL comprising the amino acid sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO : 5, SEQ ID NO: 35, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 51;
(j) Antibodies comprising: VH (XC157) comprising the amino acid sequence SEQ ID NO: 12 and VL comprising the amino acid sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO : 5, SEQ ID NO: 35, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 51;
(k) Antibodies comprising: VH (XC350) comprising the amino acid sequence SEQ ID NO: 53 and VL comprising the amino acid sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO : 5, SEQ ID NO: 35, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 51;
(l) Antibodies comprising: VH (XC351) comprising the amino acid sequence SEQ ID NO: 54 and VL comprising the amino acid sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO : 5, SEQ ID NO: 35, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 51;
(m) Antibodies comprising: VH (XC352) comprising the amino acid sequence SEQ ID NO: 55 and VL comprising the amino acid sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO : 5, SEQ ID NO: 35, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 51;
(n) Antibody comprising: VH (XC353) comprising the amino acid sequence SEQ ID NO: 56 and VL comprising the amino acid sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO : 5, SEQ ID NO: 35, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 51;
(o) Antibody comprising: VH (XC354) comprising the amino acid sequence SEQ ID NO: 55 and VL comprising the amino acid sequence selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO : 5, SEQ ID NO: 35, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 51;
(p) Antibody comprising: VL (XC151) comprising the amino acid sequence SEQ ID NO: 47 and VH comprising the amino acid sequence selected from the group consisting of: SEQ ID NO: 6, SEQ ID NO : 10, SEQ ID NO: 12, SEQ ID NO: 36, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57 ;
(q) Antibodies comprising: VL (XC153) comprising the amino acid sequence SEQ ID NO: 5 and VH comprising the amino acid sequence selected from the group consisting of: SEQ ID NO: 6, SEQ ID NO : 10, SEQ ID NO: 12, SEQ ID NO: 36, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57 ;
(r) Antibodies comprising: VL (XC154) comprising the amino acid sequence SEQ ID NO: 1 and VH comprising the amino acid sequence selected from the group consisting of: SEQ ID NO: 6, SEQ ID NO : 10, SEQ ID NO: 12, SEQ ID NO: 36, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57 ;
(s) Antibodies comprising: VL (XC346) comprising the amino acid sequence SEQ ID NO: 48 and VH comprising the amino acid sequence selected from the group consisting of: SEQ ID NO: 6, SEQ ID NO : 10, SEQ ID NO: 12, SEQ ID NO: 36, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57 ;
(t) Antibody comprising: VL (XC347) comprising the amino acid sequence SEQ ID NO: 49 and VH comprising the amino acid sequence selected from the group consisting of: SEQ ID NO: 6, SEQ ID NO : 10, SEQ ID NO: 12, SEQ ID NO: 36, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57 ;
(u) Antibodies comprising: VL (XC348) comprising the amino acid sequence SEQ ID NO: 50 and VH comprising the amino acid sequence selected from the group consisting of: SEQ ID NO: 6, SEQ ID NO : 10, SEQ ID NO: 12, SEQ ID NO: 36, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57 ;
(v) Antibodies comprising: VL (XC349) comprising the amino acid sequence SEQ ID NO: 51 and VH comprising the amino acid sequence selected from the group consisting of: SEQ ID NO: 6, SEQ ID NO : 10, SEQ ID NO: 12, SEQ ID NO: 36, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56 and SEQ ID NO: 57 ;
(w) Antibody comprising: VL (m 41A10 VH) comprising the amino acid sequence SEQ ID NO: 38 and VH comprising the amino acid sequence selected from the group consisting of: SEQ ID NO: 13, SEQ ID NO: 37, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61 and SEQ ID NO: 62;
(x) Antibodies comprising: VH (XC148) comprising the amino acid sequence SEQ ID NO: 17 and VL comprising the amino acid sequence selected from the group consisting of: SEQ ID NO: 13, SEQ ID NO : 37, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61 and SEQ ID NO: 62;
(y) Antibodies comprising: VH (XC147) comprising the amino acid sequence SEQ ID NO: 18 and VL comprising the amino acid sequence selected from the group consisting of: SEQ ID NO: 13, SEQ ID NO : 37, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61 and SEQ ID NO: 62;
(z) Antibodies comprising: VH (XC150) comprising the amino acid sequence SEQ ID NO: 63 and VL comprising the amino acid sequence selected from the group consisting of: SEQ ID NO: 13, SEQ ID NO : 37, SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61 and SEQ ID NO: 62;
(aa) an antibody comprising: a VL comprising the amino acid sequence SEQ ID NO: 37 (mouse h41A10 VL), and a VH comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 17 , SEQ ID NO: 18, SEQ ID NO: 38 and SEQ ID NO: 63;
(bb) an antibody comprising: a VL comprising the amino acid sequence SEQ ID NO: 13 (h41A10 XC142 VL), and a VH comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 38 and SEQ ID NO: 63;
(cc) an antibody comprising: a VL comprising the amino acid sequence SEQ ID NO: 58 (h41A10 XC143 VL), and a VH comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 38 and SEQ ID NO: 63;
(dd) An antibody comprising: VL (h41A10 XC144 VL) comprising the amino acid sequence SEQ ID NO: 59, and VH comprising the amino acid sequence selected from the group consisting of: SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 38 and SEQ ID NO: 63;
(ee) an antibody comprising: a VL comprising the amino acid sequence SEQ ID NO: 60 (h41A10 XC145 VL), and a VH comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 38 and SEQ ID NO: 63;
(ff) an antibody comprising: a VL comprising the amino acid sequence SEQ ID NO: 61 (h41A10 XC1446 VL), and a VH comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 38 and SEQ ID NO: 63;
(gg) an antibody comprising: a VL comprising the amino acid sequence SEQ ID NO: 62 (h41A10 XC149 VL), and a VH comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 38 and SEQ ID NO: 63;
(hh) antibodies comprising: VH (mouse 5H7 VH) comprising the amino acid sequence SEQ ID NO: 40, and VL comprising the amino acid sequence SEQ ID NO: 39;
(ii) antibodies comprising: VH comprising the amino acid sequence SEQ ID NO: 52 and VL comprising the amino acid sequence SEQ ID NO: 47;
(jj) an antibody comprising: VH comprising the amino acid sequence SEQ ID NO: 6 and VL comprising the amino acid sequence SEQ ID NO: 5;
(kk) an antibody comprising: VH comprising the amino acid sequence SEQ ID NO: 6 and VL comprising the amino acid sequence SEQ ID NO: 1;
(ll) (ii) an antibody comprising: VH comprising the amino acid sequence SEQ ID NO: 10 and VL comprising the amino acid sequence SEQ ID NO: 5;
(mm) antibodies comprising: VH comprising the amino acid sequence SEQ ID NO: 10 and VL comprising the amino acid sequence SEQ ID NO: 5; and
(nn) Antibodies comprising: VH comprising the amino acid sequence SEQ ID NO: 12 and VL comprising the amino acid sequence SEQ ID NO: 1.
E114. An antibody or antigen-binding fragment thereof that competes with any of the antibodies or antigen-binding fragments of E1 to E113 for binding to human CXCR5.
E115. An isolated nucleic acid encoding the antibody or antigen-binding fragment of any one of E1 to E114.
E116. An isolated nucleic acid molecule comprising at least one nucleic acid sequence encoding the antibody or antigen-binding fragment of any one of E1 to E109.
E117. An isolated nucleic acid molecule comprising a nucleic acid sequence selected from the group consisting of the sequences set forth in SEQ ID NO: 95, SEQ ID NO: 96, SEQ ID NO: 97, and SEQ ID NO: 98.
E118. An isolated nucleic acid molecule comprising the nucleic acid sequence set forth in SEQ ID NO: 95.
E119. An isolated nucleic acid molecule comprising the nucleic acid sequence set forth in SEQ ID NO: 96.
E120. An isolated nucleic acid molecule comprising the nucleic acid sequence set forth in SEQ ID NO: 95, and the nucleic acid sequence set forth in SEQ ID NO: 96.
E121. An isolated nucleic acid molecule comprising the nucleic acid sequence set forth in SEQ ID NO: 97.
E122. An isolated nucleic acid molecule comprising the nucleic acid sequence set forth in SEQ ID NO: 98.
E123. An isolated nucleic acid molecule comprising the nucleic acid sequence set forth in SEQ ID NO: 97, and the nucleic acid sequence set forth in SEQ ID NO: 98.
E124. An isolated nucleic acid molecule comprising the nucleic acid sequence of an insert deposited with the ATCC and having a plastid with accession number PTA-124323.
E125. An isolated nucleic acid molecule comprising the nucleic acid sequence of an insert deposited with ATCC and having a plastid with accession number PTA-124324.
E126. An isolated nucleic acid encoding VH, VL, or both of an antibody or antigen-binding fragment that specifically binds CXCR5, wherein the nucleic acid comprises: nucleic acid sequence SEQ ID NO: 95, nucleic acid sequence SEQ ID NO: 107, or both.
E127. An isolated nucleic acid encoding the heavy chain, light chain, or both of an antibody or antigen-binding fragment that specifically binds CXCR5, wherein the nucleic acid comprises: nucleic acid sequence SEQ ID NO: 97, nucleic acid sequence SEQ ID NO: 98 or both By.
E128. An isolated nucleic acid encoding a VH of an antibody or antigen-binding fragment that specifically binds CXCR5, wherein the nucleic acid includes a nucleic acid sequence deposited with an ATCC and having a plastid with accession number PTA-124323.
E129. An isolated nucleic acid encoding a VL of an antibody or antigen-binding fragment that specifically binds CXCR5, wherein the nucleic acid includes a nucleic acid sequence deposited with an ATCC and having an insert of plastid with accession number PTA-124324.
E130. An isolated nucleic acid encoding VL and VH of an antibody or antigen-binding fragment that specifically binds to CXCR5, wherein the nucleic acid includes a nucleic acid sequence deposited with an ATCC and having a plastid with accession number PTA-124324, ATCC has the nucleic acid sequence of the insert with the registration number PTA-124323 plastid.
E131. A vector comprising the nucleic acid molecule according to any one of E115 to E130.
E132. A host cell comprising the nucleic acid molecule of any one of E115 to E130, or the vector of E131.
E133. The host cell as E132, wherein the cell is a mammalian cell.
E134. A host cell such as E133, wherein the host cell is a CHO cell, COS cell, HEK-293 cell, NS0 cell, PER.C6® cell, or Sp2.0 cell.
E135. The host cell of any one of E132 to 134, wherein the host cell lacks functional α-1,6-fucosyltransferase (FUT8).
E136. The host cell of any one of E132 to 135, wherein the cell does not express functional α-1,6-fucosyltransferase.
E137. The host cell of any one of E132 to 136, wherein the cell lacks the FUT8 gene encoding a functional enzyme.
E138. The host cell of any one of E132 to 137, wherein the cell lacks coding functionality FUT8 Gene gene.
E139. The host cell of any one of E132 to 139, wherein the cell line is a Potelligent® CHOK1SV cell or a Lec13 CHO cell.
E140. Such as the host cell of E139, wherein the cell line is Potelligent® CHOK1SV cell.
E141. A method for preparing an antibody or antigen-binding fragment thereof, which comprises culturing a host cell according to any one of E1 to E114 under conditions in which the antibody or antigen-binding fragment is expressed by the host cell.
E142. The method of E141 further comprises isolating the antibody or antigen-binding fragment thereof.
E143. A method for preparing afucosylated anti-CXCR5 antibody or antigen-binding fragment thereof, the method comprising cultivating a host cell comprising a nucleic acid molecule according to any one of E115 to E130 or a vector such as E131, wherein the host cell lacks function性 FUT8.
E144. The method as E143, wherein the host cell is a Potelligent® CHOK1SV cell.
E145. An isolated antibody or antigen-binding fragment thereof, which is prepared using any one of methods E141 to E114.
E146. The isolated antibody or antigen-binding fragment thereof according to any one of E1 to E114, wherein the antibody or antigen-binding fragment thereof is defucosylated.
E147. Defucosylated antibody or antigen-binding fragment thereof such as E146, wherein the defucosylated antibody or antigen-binding fragment thereof is compared to an antibody or antigen-binding fragment thereof that is otherwise identical to fucosylated The fragments exhibit enhanced antibody-dependent cytotoxicity (ADCC).
E148. Defucosylated antibody or antigen-binding fragment thereof such as E146, wherein the defucosylated antibody or antigen-binding fragment thereof is compared with an antibody or antigen-binding fragment thereof that is otherwise the same as fucosylated , Showing about 2 times, about 5 times, about 7 times, about 10 times, about 20 times, about 30 times, about 40 times, about 50 times, about 60 times, about 70 times, about 80 times, about 80 times, about ADCC of 90 times, about 100 times, about 110 times, about 120 times, about 130 times, about 140 times, and about 143 times.
E149. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of E1 to E114 and E145 to E148 and a pharmaceutically acceptable carrier or excipient.
E150. A pharmaceutical composition as in E149, wherein the antibody or antigen-binding fragment thereof is defucosylated.
E151. A method of reducing the activity of CXCR5, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody or antigen-binding fragment thereof such as any one of E1 to E114 and E145 to E148, or any one such as E149 and E150 Pharmaceutical composition.
E152. A method for treating inflammatory diseases, which comprises administering to a subject in need thereof a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any one of E1 to E114 and E145 to E148, or any one according to E149 to E150 Pharmaceutical composition.
E153. A method of treating an individual in need of immunosuppression, comprising administering to an individual in need a therapeutically effective amount of an antibody or antigen-binding fragment thereof such as any one of E1 to E114 and E145 to E148, or any one such as E149 and E150 A pharmaceutical composition.
E154. A method of treating an autoimmune disease, disorder or condition, which comprises administering to a subject in need thereof a therapeutically effective amount of an antibody or antigen-binding fragment thereof such as any one of E1 to E114 and E145 to E148, or such as E149 And the pharmaceutical composition of any one of E150.
E155. A method for reducing the number of cells expressing CXCR5 in an individual in need thereof, the method comprising administering to the individual a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any one of E1 to E114 and E145 to E148, or such as E149 and The pharmaceutical composition of any one of E150.
E156. The method as E155, wherein the cells express CXCR5 on their surface.
E157. The method as in E156, wherein the cells are B cells and Tfh-like cells.
E158. The method according to any one of E E151 to E157, wherein the system is human.
E159. The method according to any one of E151 to E158, which comprises intravenously administering the antibody or antigen-binding fragment or pharmaceutical composition thereof.
E160. The method according to any one of E151 to E158, which comprises subcutaneous administration of the antibody or antigen-binding fragment or pharmaceutical composition thereof.
E161. The method according to any one of E151 to E160, wherein the antibody or its antigen-binding fragment or pharmaceutical composition is administered twice a week, once a week, once every two weeks, once every three weeks, once every four weeks, every five weeks Once, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, twice a month, once a month, once every two months, once every three months, once every four months , Once every May, once every June, once every July, once every August, once every September, once every October, once every November or once every December.
E162. A method for reducing the number of CXCR5 + cells in a sample, the method comprising allowing the cell to bind to the antibody or antigen-binding fragment of any one of E1 to E114 and E145 to E148, or the pharmaceutical composition of any one of E149 and E150 contact.
E163. An antibody or antigen-binding fragment thereof according to any one of E1 to E114 and E145 to E148, or a pharmaceutical composition according to any one of E149 and E150, which is used as a medicament.
E164. The antibody or antigen-binding fragment of any one of E1 to E114 and E145 to E148, or the pharmaceutical composition of any one of E149 and E150, is used to reduce CXCR5 activity in an individual.
E165. An antibody or antigen-binding fragment thereof according to any one of E1 to E114 and E145 to E148, or a pharmaceutical composition according to any one of E149 and E150, which is used to treat an individual in need of immunosuppression.
E166. The antibody or antigen-binding fragment of any one of E1 to E114 and E145 to E148, or the pharmaceutical composition of any one of E149 and E150, which is used to treat an autoimmune disease, disorder or condition in an individual .
E167. Use of an antibody or antigen-binding fragment thereof according to any one of E1 to E114 and E145 to E148 in the manufacture of a medicament for the treatment of immune diseases, disorders or conditions.
E168. Use of a pharmaceutical composition as in any of E149 and E150 in the manufacture of a medicament for the treatment of immune diseases, disorders or conditions.
E169. A method for treating a medical condition, which comprises administering to a subject in need thereof a therapeutically effective amount of an antibody or antigen-binding fragment thereof such as any one of E1 to E114 and E145 to E148, or any one such as E149 and E150 Pharmaceutical composition.
E170. A method as in E169, wherein the condition is selected from the group consisting of: inflammatory reactions, such as inflammatory skin diseases, including psoriasis and dermatitis (eg, atopic dermatitis); dermatomyositis; systemic scleroderma and sclerosis Syndrome; reactions associated with inflammatory bowel disease (such as Crohn's disease and ulcerative colitis); respiratory distress syndrome (including adult respiratory distress syndrome; ARDS); dermatitis; meningitis; encephalitis; Uveitis; colitis; gastritis; glomerulonephritis; allergic constitutions such as eczema and asthma and other conditions involving T cell infiltration and chronic inflammatory reactions; atherosclerosis; leukocyte adhesion deficiency; rheumatoid Arthritis; systemic lupus erythematosus (SLE); diabetes (such as type 1 diabetes or insulin-dependent diabetes); multiple sclerosis; Reynaud's syndrome; autoimmune thyroiditis; allergic encephalomyelitis ; Sjogren's syndrome; juvenile-onset diabetes; and mediated by cytokines and T lymphocytes commonly found in tuberculosis, sarcomatosis, polymyositis, granuloma, and vasculitis Immune reactions related to acute and delayed allergic reactions; Wegener's disease; Pernicious anemia (Addison's disease); Diseases involving leukocyte exudation; Central nervous system (CNS) inflammation Disorders; multiple organ damage syndrome; hemolytic anemia (including but not limited to cryoglobulinemia or Qom's-positive anemia); myasthenia gravis; disease mediated by antigen-antibody complex; anti-glomerular basement membrane disease; Antiphospholipid syndrome; allergic neuritis; Graves'disease; Lambert-Eaton myasthenic syndrome; bullous pemphigoid; pemphigus; autoimmune polyendocrine Lesions; White spot disease; Reiter's disease; Zombie syndrome; Bechet disease; Giant cell arteritis; Immune complex nephritis; IgA nephropathy; IgM polyneuropathy; Immune thrombocytopenia Purpura (ITP) or autoimmune thrombocytopenia and autoimmune hemolytic disease; Hashimoto's thyroiditis; autoimmune hepatitis; autoimmune hemophilia; autoimmune lymphoproliferative syndrome (ALPS); autoimmune grapes Membranous retinitis; Guillain-Barre syndrome; Goodpasture's syndrome; mixed connective tissue disease; autoimmune-related infertility; polyarteritis nodosa; alopecia areata; Idiopathic mucus edema; graft-versus-host disease; muscular dystrophy (Duchen's type, Becker type, muscular tone type, limb band type, facial (Scapula arm type, congenital type, ophthalmopharyngeal type, distal type, Ai-De type) and control the proliferation of CXCR5 cancer cells, such as pancreatic cancer, colon cancer, bladder cancer, T cell leukemia and B cell leukemia.
E171. A method for detecting CXCR5 in a sample, tissue, or cell using the antibody or antigen-binding fragment of any one of E1 to E114 and E145 to E148, or the pharmaceutical composition of any one of E149 and E150, comprising The sample, tissue or cell is contacted with the antibody and the antibody is detected.
E172. An isolated antibody that specifically binds CXCR5, or an antigen-binding fragment thereof, wherein the anti-system is at least one antibody selected from the group consisting of:
(a) an antibody comprising: CDR-L1 comprising the amino acid sequence SEQ ID NO: 2; CDR-L2 comprising the amino acid sequence SEQ ID NO: 3; CDR-L2 comprising the amino acid sequence SEQ ID NO: 4 CDR-L3; CDR-H1 comprising the amino acid sequence SEQ ID NO: 7; CDR-H2 comprising the amino acid sequence SEQ ID NO: 8; and CDR-H3 comprising the amino acid sequence SEQ ID NO: 9 ;
(b) an antibody comprising: CDR-L1 comprising the amino acid sequence SEQ ID NO: 2; CDR-L2 comprising the amino acid sequence SEQ ID NO: 3; CDR-L2 comprising the amino acid sequence SEQ ID NO: 4 CDR-L3; CDR-H1 comprising the amino acid sequence SEQ ID NO: 7; CDR-H2 comprising the amino acid sequence SEQ ID NO: 8; and CDR-H3 comprising the amino acid sequence SEQ ID NO: 11 ;
(c) an antibody comprising: CDR-L1 comprising the amino acid sequence SEQ ID NO: 14; CDR-L2 comprising the amino acid sequence SEQ ID NO: 15; CDR-L2 comprising the amino acid sequence SEQ ID NO: 16 CDR-L3; CDR-H1 comprising the amino acid sequence SEQ ID NO: 19; CDR-H2 comprising the amino acid sequence SEQ ID NO: 20; and CDR-H3 comprising the amino acid sequence SEQ ID NO: 21 ;
(d) Antibodies containing the following: VL containing the amino acid sequence encoded by the insert deposited with ATCC and having the accession number PTA-124324; VH of the amino acid sequence encoded by the plastid insert;
(e) Antibodies comprising: VL comprising the amino acid sequence SEQ ID NO: 1 and VH comprising the amino acid sequence SEQ ID NO: 6;
(f) an antibody comprising: VL comprising the amino acid sequence SEQ ID NO: 13 and VH comprising the amino acid sequence SEQ ID NO: 18;
(g) an antibody comprising: VL comprising the amino acid sequence SEQ ID NO: 47, and VH comprising the amino acid sequence SEQ ID NO: 52;
(h) antibodies comprising: VL comprising the amino acid sequence SEQ ID NO: 5 and VH comprising the amino acid sequence SEQ ID NO: 6;
(i) antibodies comprising: VL comprising the amino acid sequence SEQ ID NO: 5 and VH comprising the amino acid sequence SEQ ID NO: 10;
(j) an antibody comprising: VL comprising the amino acid sequence SEQ ID NO: 13 and VH comprising the amino acid sequence SEQ ID NO: 17;
(k) antibodies comprising: VL comprising the amino acid sequence SEQ ID NO: 1 and VH comprising the amino acid sequence SEQ ID NO: 12;
(l) an antibody comprising: an LC comprising the amino acid sequence SEQ ID NO: 22, and an HC comprising the amino acid sequence SEQ ID NO: 23;
(m) Antibodies comprising: LC comprising the amino acid sequence SEQ ID NO: 24, and HC comprising the amino acid sequence SEQ ID NO: 25;
(n) Antibodies comprising: LC comprising the amino acid sequence SEQ ID NO: 26, and HC comprising the amino acid sequence SEQ ID NO: 27;
(o) an antibody comprising: an LC comprising the amino acid sequence SEQ ID NO: 28, and an HC comprising the amino acid sequence SEQ ID NO: 29;
(p) antibodies comprising: VL encoded by nucleic acid sequence SEQ ID NO: 95, and VH encoded by nucleic acid sequence SEQ ID NO: 96; and
(q) Antibodies comprising: LC encoded by nucleic acid sequence SEQ ID NO: 97, and HC encoded by nucleic acid sequence SEQ ID NO: 98.
E173. An isolated antibody or antigen-binding fragment thereof that specifically binds CXC-chemokine receptor 5 (CXCR5), wherein the antibody or antigen-binding fragment thereof is at least one antibody selected from the group consisting of:
(a) The following antibody or antigen-binding fragment thereof: which binds to the hCXCR5 epitope comprising leucine at amino acid residue number 11 according to the amino acid sequence SEQ ID NO: 32, but does not bind to it The residue is not the epitope of leucine;
(b) The following antibody or antigen-binding fragment thereof: which binds to the hCXCR5 epitope comprising leucine at amino acid residue number 11 according to the amino acid sequence SEQ ID NO: 32, but does not bind to it The residue is the epitope of threonine;
(c) The following antibody or antigen-binding fragment thereof: which binds to the hCXCR5 epitope containing aspartic acid at amino acid residue number 22 according to the amino acid sequence SEQ ID NO: 32, but does not bind Where the residue is not the epitope of aspartic acid;
(d) The following antibody or antigen-binding fragment thereof: which binds to the hCXCR5 epitope containing aspartic acid at amino acid residue number 22 according to the amino acid sequence SEQ ID NO: 32, but does not bind Where the residue is the epitope of alanine;
(e) The following antibody or antigen-binding fragment thereof: the binding comprises leucine at amino acid residue number 11 according to the amino acid sequence SEQ ID NO: 32 and at amino acid residue number 22 HCXCR5 epitope of aspartic acid, but does not bind to the epitope in which the leucine is substituted with threonine and / or the aspartic acid is substituted with alanine;
(f) The following antibody or antigen-binding fragment thereof: which binds hCXCR5 or a fragment thereof comprising leucine at amino acid residue number 11 according to the amino acid sequence SEQ ID NO: 32, but does not bind to it The residue is not hCXCR5 of leucine or a fragment thereof;
(g) The following antibody or antigen-binding fragment thereof: which binds to hCXCR5 or a fragment thereof comprising leucine at amino acid residue number 11 according to the amino acid sequence SEQ ID NO: 32, but does not bind to it The residue is hCXCR5 of threonine or a fragment thereof;
(h) The following antibody or antigen-binding fragment thereof: which binds hCXCR5 or its fragment comprising aspartic acid at amino acid residue number 22 according to the amino acid sequence SEQ ID NO: 32, but does not bind Where the residue is not hCXCR5 of aspartic acid or a fragment thereof;
(i) The following antibody or antigen-binding fragment thereof: which binds hCXCR5 or its fragment comprising aspartic acid at amino acid residue number 22 according to the amino acid sequence SEQ ID NO: 32, but does not bind Wherein the residue is hCXCR5 of alanine or a fragment thereof; and
(j) The following antibody or antigen-binding fragment thereof: the binding comprises the leucine acid at amino acid residue number 11 and the amino acid residue number 22 according to the amino acid sequence SEQ ID NO: 32 HCXCR5 of aspartic acid or a fragment thereof, but not hCXCR5 or a fragment thereof in which the leucine is substituted with threonine and / or the aspartic acid is substituted with alanine.
E174. An isolated antibody or antigen-binding fragment thereof that specifically binds CXC-chemokine receptor 5 (CXCR5), wherein the antibody or antigen-binding fragment thereof is at least one antibody selected from the group consisting of:
(a) The following antibody or antigen-binding fragment thereof, which binds hCXCR5 expressed on human B cells with an apparent affinity of EC50 of about 6.60 pM and a standard deviation of about ± 2.33 pM;
(b) The following antibody or antigen-binding fragment thereof, which binds hCXCR5 expressed on human circulating follicular T helper-like cells with an apparent affinity of EC50 of about 5.89 pM and a standard deviation of about ± 1.40 pM;
(c) The following antibody or antigen-binding fragment thereof, which binds hCXCR5 expressed on human follicular T helper (Tfh) cells with an apparent affinity of EC50 of about 10.6 pM;
(d) The following antibody or antigen-binding fragment thereof, which binds cynoCXCR5 expressed on cynomolgus macaque B cells with an apparent affinity of EC50 of about 1.32 pM;
(e) The following antibody or antigen-binding fragment thereof, which binds cynoCXCR5 expressed on cynomolgus monkey Tfh-like cells with an apparent affinity of EC50 of about 10.5 pM;
(f) The following antibody or antigen-binding fragment thereof: It antagonizes CXCR5-CXCL13 signaling in cAMP report analysis with an EC50 of approximately 961 pM;
(g) The following antibody or antigen-binding fragment thereof: which exhibits ADCC activity on human B cells expressing hCXCR5 with an EC50 of about 2.01 pM and a standard deviation of about ± 2.28 pM;
(h) The following antibody or antigen-binding fragment thereof: It exhibits ADCC activity on human Tfh-like cells expressing hCXCR5 with an EC50 of about 4.28 pM and a standard deviation of about ± 2.88 pM;
(i) The following antibodies or antigen-binding fragments thereof: exhibit ADCC activity of human Tfh cells expressing hCXCR5 with an EC50 of about 0.11 pM;
(j) The following antibody or antigen-binding fragment thereof: which exhibits ADCC activity of EC50 of about 15.3 pM and a standard deviation of about ± 11.7 pM on cynoCXCR5 cynomolgus monkey B cells expressing cynoCXCR5;
(k) The following antibody or antigen-binding fragment thereof: which binds hCXCR5 but does not detectably bind to the human chemotactic receptor CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CMKLR1 , CXCR3R1, CXCR1, CXCR2, CXCR3, CXCR4, CXCR6, CXCR7 and XCR1;
(l) Antibodies or antigen-binding fragments that inhibit the binding of CXCR5 to CXCL13;
(m) Antibodies or antigen-binding fragments that bind to CXCR5 + human B cells with an apparent affinity of EC50 less than about 26 pM, but do not bind to cells expressing CXCR5 mouse, rat, or rabbit heterologs;
(n) Antibodies or antigen-binding fragments that antagonize the inhibition of cXCL13 released by cAMP triggered by forskolin;
(o) antibodies or antigen-binding fragments that trigger ADCC of CXCR5 expressing cells in human donor and cynomolgus monkey PBMC and human donor TMC;
(p) The following antibodies or antigen-binding fragments: which bind to human CXCR5 but not to human chemokine receptors CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CMKLR1, CXCR3R1, CXCR1 , CXCR2, CXCR3, CXCR4, CXCR6, CXCR7 or XCR1;
(q) antibodies or antigen-binding fragments that deplete B cells in peripheral blood;
(r) antibodies or antigen-binding fragments that deplete Tfh-like cells in peripheral blood;
(s) antibodies or antigen-binding fragments that deplete bona fide Tfh cells in the spleen; and
(t) Antibodies or antigen-binding fragments that impair the humoral immune memory response.
E175. The antibody of any one of E172 to E174, wherein the antibody or antigen-binding fragment thereof exhibits at least one of the following biological activities:
(a) Binding CXCR5 + human B cells with an apparent affinity of EC50 less than about 26 pM, but not cells expressing CXCR5 mouse, rat, or rabbit xenologs;
(b) antagonize CXCL13 inhibition of cAMP release triggered by forskolin;
(c) Trigger ADCC of CXCR5 expressing cells in human donor and cynomolgus monkey PBMC and human donor TMC;
(d) Binding to human CXCR5 but not to human chemotactic receptor CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CMKLR1, CXCR3R1, CXCR1, CXCR2, CXCR3, CXCR4, CXCR6, CXCR7 or XCR1;
(e) Depletion of B cells in the peripheral blood;
(f) deplete Tfh-like cells in the peripheral blood;
(g) depleting bona fide Tfh cells in the spleen; or
(h) Impair humoral immune memory response.
E176. The antibody of any one of E172 to E175, wherein the antibody or antigen-binding fragment thereof is defucosylated.
E177. An isolated nucleic acid encoding the antibody or antigen-binding fragment of any one of E172 to E176.
E178. An isolated nucleic acid encoding VH, VL, or both of an antibody or antigen-binding fragment that specifically binds CXCR5, wherein the nucleic acid comprises: nucleic acid sequence SEQ ID NO: 95, nucleic acid sequence SEQ ID NO: 96, or both.
E179. An isolated nucleic acid encoding the heavy chain, light chain, or both of an antibody or antigen-binding fragment that specifically binds CXCR5, wherein the nucleic acid comprises: nucleic acid sequence SEQ ID NO: 97, nucleic acid sequence SEQ ID NO: 98 or both By.
E180. An isolated nucleic acid encoding VH, VL, or both of an antibody or antigen-binding fragment that specifically binds CXCR5, wherein the nucleic acid contains the nucleic acid sequence of an insert deposited with ATCC and having a plastid with registration number PTA-124323 The nucleic acid sequence of an insert deposited with ATCC and having a plastid with accession number PTA-124324, or both.
E181. A vector comprising the nucleic acid according to any one of E177 to E180.
E182. A host cell comprising a vector such as E181.
E183. A host cell such as E182, wherein the host cell line is selected from the group consisting of mammalian cells consisting of CHO cells, COS cells, HEK-293 cells, NS0 cells, PER.C6® cells or Sp2.0 cells.
E184. A host cell like E183, where the cell lacks functional α-1,6-fucosyltransferase (FUT8).
E185. Such as the host cell of E184, wherein the cell line is Potelligent® CHOK1SV cell or Lec13 CHO cell.
E186. A method for preparing an antibody, or antigen-binding fragment thereof, under conditions where the antibody or antigen-binding fragment thereof is expressed by Potelligent® CHOK1SV cells such as E185 and is defucosylated, which includes the host cell.
E187. The method of E186, further comprising isolating the antibody or antigen-binding fragment thereof.
E188. The defucosylated antibody of E186, or an antigen-binding fragment thereof, wherein the antibody exhibits enhanced ADCC activity when compared to an antibody or antigen-binding fragment thereof that is otherwise fucosylated.
E189. A pharmaceutical composition comprising the antibody or antigen-binding fragment of any one of E172 to E176 and E188, and a pharmaceutically acceptable carrier or excipient.
E190. For example, the antibody or antigen-binding fragment of any one of E172 to E176, E188, and the pharmaceutical composition of E189, which are used for the treatment of immune diseases, disorders or conditions.
E191. An antibody or antigen-binding fragment thereof according to any one of E172 to E176, E188, or a pharmaceutical composition such as E189, for the treatment of immune diseases, disorders, or conditions.
E192. A method for treating or preventing a CXCR5-mediated immune disease, disorder or condition in a human individual in need, the method comprising administering to the individual an effective amount of a pharmaceutical composition such as E189, wherein the disease, disorder or The condition is selected from the group consisting of: inflammatory reactions, such as inflammatory skin diseases, including psoriasis and dermatitis (eg atopic dermatitis); dermatomyositis; systemic scleroderma and sclerosis; Responses such as Crohn's disease and ulcerative colitis; respiratory distress syndrome (including adult respiratory distress syndrome; ARDS); dermatitis; meningitis; encephalitis; uveitis; colitis; gastritis ; Glomerulonephritis; allergic constitutions, such as eczema and asthma, and other conditions involving T cell infiltration and chronic inflammatory reactions; atherosclerosis; leukocyte adhesion deficiency; rheumatoid arthritis (RA); systemic Lupus erythematosus (SLE); diabetes (eg, type I diabetes or insulin-dependent diabetes); multiple sclerosis; Reynaud's syndrome; autoimmune thyroiditis; allergic encephalomyelitis; Sugren's Syndrome (Sjogren's syndrome); juvenile-onset diabetes; and acute and delayed mediated by interleukins and T lymphocytes commonly found in tuberculosis, sarcomatosis, polymyositis, granuloma, and vasculitis Immune reactions related to allergic reactions; Wegener's disease; Pernicious anemia (Addison's disease); Diseases involving leukocyte exudation; Inflammatory disorders of the central nervous system (CNS); Multiple organ damage Syndromes; hemolytic anemia (including but not limited to cryoglobulinemia or Qom's-positive anemia); myasthenia gravis; diseases mediated by antigen-antibody complex; anti-glomerular basement membrane disease; antiphospholipid syndrome; allergy Neuritis; Graves'disease; Lambert-Eaton myasthenic syndrome; bullous pemphigoid; pemphigus; autoimmune multiple endocrine lesions; leukoplakia; Reiter's disease; stiffness syndrome; Bechet disease; giant cell arteritis; immune complex nephritis; IgA nephropathy; IgM polyneuropathy; immune thrombocytopenic purpura (ITP) or Autoimmune thrombocytopenia and autoimmune hemolytic disease; Hashimoto's thyroiditis; autoimmune hepatitis; autoimmune hemophilia; autoimmune lymphoproliferative syndrome (ALPS); autoimmune uveitis retinitis; grid -Guillain-Barre syndrome; Goodpasture's syndrome; mixed connective Histopathology; autoimmunity-related infertility; polyarteritis nodosa; alopecia areata; idiopathic mucus edema; graft-versus-host disease; muscular dystrophy (Duchen's type, Becker type, muscular tone type, limb (Band type, scapular arm type, congenital type, oropharyngeal type, distal type, Ai-De type) and cancer cells that control CXCR5, such as pancreatic cancer, colon cancer, bladder cancer, T cell leukemia and B cells Proliferation of leukemia.
E193. The method as E192, wherein the disease is SLE or rheumatoid arthritis.
E194. Use of an antibody or antigen-binding fragment thereof according to any one of E172 to E176 or E188 in the manufacture of a medicament for the treatment of immune diseases, disorders or conditions.
E195. A method for detecting CXCR5 in a sample, tissue, or cell using the antibody or antigen-binding fragment of any one of E172 to E176, which comprises contacting the sample, tissue, or cell with the antibody and detecting the antibody.
E196. A method of reducing the biological activity of CXCR5 in an individual in need thereof, the method comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof such as any one of E172 to E176 or E188, or a pharmaceutical composition such as E189.
E197. The method of E196, wherein the antibody mediates the depletion of at least one cell selected from the group consisting of CXCR5 expressing: Tfh cells in the spleen, B cells in peripheral blood, and Tfh-like cells in peripheral blood.
E198. A method of inhibiting a humoral immune response in an individual in need thereof, the method comprising administering a therapeutically effective amount of an antibody or antigen-binding fragment thereof as in any of E172 to E176 or E188, or a pharmaceutical composition as in E189.
E199. The method of E198, wherein the antibody mediates depletion of at least one cell selected from the group consisting of CXCR5 expressing: Tfh cells in the spleen, B cells in peripheral blood, and Tfh-like cells in peripheral blood.

Related application

This application claims the priority of US No. 62 / 593,830 filed on December 1, 2017 and US No. 62 / 732,985 filed on September 18, 2018, the contents of which are incorporated herein by reference in their entirety.
Sequence Listing

This specification is further incorporated by reference into the sequence listing provided with it on November 28, 2018. According to 37 CFR § 1.52 (e) (5), the sequence list text file identified as PC72320A_Seq_Listing_ST25.txt is 112,891 bytes and was created on November 15, 2018. The sequence listing applied for electronically with it does not extend beyond the scope of the specification and therefore does not contain new topics.
Parties that have reached a joint research statement

The currently claimed invention is completed by or in the name of the following parties who have reached a joint research agreement. The joint research agreement takes effect on or before the date the claimed invention is completed, and the claimed invention is completed as a result of activities carried out within the scope of the joint research agreement. The parties to the joint research agreement represent THE REGENTS OF THE UNIVERSITY OF CALIFORNIA and PFIZER INC of SAN DIEGO CAMPUS.

This article discloses antibodies that specifically bind to CXCR5 and, in addition, antibodies that antagonize CXCR5 activity or interact with CXCL13. Provided are methods for preparing CXCR5 antibodies, compositions containing these antibodies, and methods for using these antibodies.

Provide fucosylated and defucosylated antibodies that bind CXCR5. In some embodiments, heavy and light chains of defucosylated antibodies capable of forming antibodies that bind to CXCR5 are also provided. In some embodiments, defucosylated antibody heavy and light chains are provided that include one or more specific complementarity determining regions (CDRs). In some embodiments, the defucosylated anti-CXCR5 antibody has varying effector functions. In some embodiments, the antibodies of the invention have enhanced ADCC activity relative to fucosylated anti-CXCR5 antibodies that are otherwise identical to the invention.

A polynucleotide encoding an antibody or antigen-binding fragment thereof that binds CXCR5 is provided. Polynucleotides encoding antibody heavy or light chains are also provided. Provide host cells that express fucosylated and / or defucosylated anti-CXCR5 antibodies. Provides treatment methods for antibodies to CXCR5 using defucosylated and fucosylated. Such methods include, but are not limited to, methods of treating diseases (including but not limited to inflammatory diseases and immune diseases) associated with or mediated by CXCR5 performance and / or binding to CXCL13.

Some of the headings used herein are for organizational purposes only and should not be construed as limiting the subject matter.

All references cited herein, including patent applications, patent publications, and Genbank deposit numbers, are incorporated by reference, as if each individual reference was specifically and individually indicated to be incorporated by reference in its entirety Medium.

The techniques and procedures described or cited herein are generally well-understood and are generally employed by those skilled in the art using conventional methods, such as the widely adopted methods described in: Sambrook et al., Molecular Cloning: A Laboratory Manual 3rd Edition (2001) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (ed by FM Ausubel et al., (2003)); METHODS IN ENZYMOLOGY series (Academic Press, Inc.): PCR 2 : A PRACTICAL APPROACH (MJ MacPherson, BD Hames and GR Taylor (1995)), Harlow and Lane (1988) ANTIBODIES, A LABORATORY MANUAL and ANIMAL CELL CULTURE (RI Freshney (1987)); Oligonucleotide Synthesis (MJ Gait) , 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (edited by JE Cellis, 1998) Academic Press; Animal Cell Culture (edited by RI Freshney, 1987); Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture Laboratory Procedures (edited by A. Doyle, JB Griffiths and DG Newell, 1993-8) J. Wiley and Sons; Handbook of Experimental Immunology (edited by DM Weir and CC Blackwell); Gene Transfer Vectors for Mammalian Cells (JM Mil ler and MP Calos, 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., 1994); Current Protocols in Immunology (JE Coligan et al., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999 ); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: A Practical Approach (Edited by D. Catty, IRL Press, 1988-1989); Monoclonal Antibodies: A Practical Approach (P Edited by Shepherd and C. Dean, Oxford University Press, 2000); Using Antibodies: A Laboratory Manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999)); The Antibodies (Edited by M. Zanetti and JD Capra , Harwood Academic Publishers, 1995); and Cancer: Principles and Practice of Oncology (Edited by VT DeVita et al., JB Lippincott Company, 1993); and its updated version.

Fucosylated or defucosylated CXCR5 antibodies can be used to prevent, treat, and / or ameliorate diseases, disorders, or conditions caused by and / or associated with CXCR5 activity. Such diseases, disorders or conditions include, but are not limited to, inflammatory reactions, such as systemic lupus erythematosus (SLE); chronic inflammatory reactions; atherosclerosis; leukocyte adhesion deficiency; rheumatoid arthritis; diabetes (e.g. I Type 2 diabetes or insulin-dependent diabetes); multiple sclerosis; Raynaud's syndrome; autoimmune thyroiditis; allergic encephalomyelitis; Huguelin's syndrome; juvenile-onset diabetes; and usually tuberculosis, sarcoma , Polymyositis, granuloma and vasculitis, the immune response related to acute and delayed allergic reactions mediated by interleukins and T lymphocytes; Wegener's disease; pernicious anemia (Addison's Disease); diseases involving leukocyte exudation; inflammation of the central nervous system (CNS); multiple organ damage syndrome; hemolytic anemia (including but not limited to cryoglobulinemia or Qom's-positive anemia); myasthenia gravis; antigen -Antibody complex-mediated diseases; anti-glomerular basement membrane disease; anti-phospholipid syndrome; allergic neuritis; Graves'disease; Lambert-Eton muscle weakness syndrome; bullous pemphigoid; pemphigus; Autoimmune multiple endocrine lesions; white spot disease; Wright's disease; stiffness syndrome; Behcet's disease; giant cell arteritis; immune complex nephritis; IgA nephropathy; IgM polyneuropathy; ) Or autoimmune thrombocytopenia and autoimmune hemolytic disease; Hashimoto's thyroiditis; autoimmune hepatitis; autoimmune hemophilia; autoimmune lymphoproliferative syndrome (ALPS); autoimmune uveitis retinitis ; Gerard-Barr's syndrome; Goodbold's syndrome; mixed connective tissue disease; autoimmune-related infertility; polyarteritis nodosa; alopecia areata; idiopathic mucus edema; graft-versus-host disease; muscle Atrophy (Duchen's type, Becker type, muscle tone type, limb band type, scapular arm type, congenital type, ophthalmopharyngeal type, distal type, Aide type) and cancer cells that control CXCR5 For example, the proliferation of pancreatic cancer, colon cancer, bladder cancer, T-cell leukemia, and B-cell leukemia is understood by those skilled in the art in consideration of the teachings disclosed herein.
I. Definition

The present invention can be more easily understood with reference to the implementation of the following exemplary embodiments of the present invention and the examples included therein.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification (including definitions) will control.

In addition, unless the context requires otherwise or otherwise expressly stated, singular terms shall include the plural and plural terms shall include the singular.

It should be understood that the aspects and embodiments of the invention described herein include "consisting of aspects and embodiments" and / or "mainly consisting of aspects and embodiments." Unless otherwise specified, as used herein, the singular forms "a" and "the" include plural references.

In this application, the use of "or" means "and / or" unless explicitly described or understood by those skilled in the art. In the case of multiple sub-items, the use of "or" refers to more than one of the aforementioned independent items or sub-items.

"About" or "approximately" when used in combination with a measurable digital variable refers to the indicator value of the variable and within the experimental error of the indicator value (such as within the 95% confidence interval of the average) or within 10 percentage points of the indicator All values of the variable (whichever is greater). The numerical range includes the numbers that define the range.

Although the numerical ranges and parameters that illustrate the broad scope of the invention are approximate, the numerical values set forth in particular embodiments should be reported as accurately as possible. However, any numerical value inherently contains certain errors necessarily caused by the standard deviation found in its corresponding test measurement. Furthermore, all ranges disclosed herein should be understood to cover any and all subranges subsumed therein. For example, the stated range of "1 to 10" should be considered to include any and all subranges between the minimum value 1 and the maximum value 10 (inclusive); that is, start with a minimum value of 1 or greater (e.g. 1 to 6.1) and all subranges ending with a maximum value of 10 or less (eg 5.5 to 10).

Throughout this specification and the scope of patent applications, the wording "comprise" or variations such as "comprises" or "comprising" should be understood to mean including the integer or group of integers but Does not exclude any other integer or group of integers. Unless the context requires otherwise, singular terms shall include pluralities and plural terms shall include the singular. Any examples after the terms "e.g." or "for example" are not meant to be exhaustive or restrictive.

It should be understood that embodiments anywhere in this document are described in the language "comprising", or similar embodiments described in terms "consisting of" and / or "consisting essentially of" are also provided.

When the aspects or embodiments of the present invention are described in terms of Markush groups or other alternative groups, the present invention covers not only all groups listed as a whole, but also members and masters of independent groups All possible subgroups of the group, and also covers the main group lacking one or more group members. The invention also contemplates the explicit exclusion of one or more of any group members in the claimed invention.

It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. In this specification and subsequent patent applications, multiple terms will be cited, and these terms should be defined to have the following meanings.

The term "isolated molecule" (where the molecule is a polypeptide, polynucleotide or antibody or fragment thereof) refers to a molecule that, by virtue of its source or derived source (1), is not associated with its natural counterpart in its natural state Relevant components are combined; (2) is substantially free of other molecules from the same species; (3) is expressed by cells from different species, or (4) does not appear in nature. Therefore, molecules that have been chemically synthesized or expressed in a cell system different from cells of natural origin will be "isolated" from their naturally-related components. Using purification techniques well known in the art, the molecules can also be substantially free of naturally related components by separation. Molecular purity or homogeneity can be analyzed by various methods well known in the art. For example, the purity of a polypeptide sample can be analyzed using polyacrylamide gel electrophoresis and gel staining to visualize the polypeptide using techniques well known in the art. For some purposes, higher resolution can be provided by using HPLC or other well-known methods used in the art for purification.

As used herein, "substantially pure" means the main species present in the target species lineage (ie, it is more abundant in the composition than any other individual species in terms of moles), and is preferably substantially pure. It is a composition in which the target species (such as glycoproteins, including antibodies or receptors) accounts for at least about 50% (in moles) of all macromolecular species present. Generally speaking, a substantially pure composition will comprise more than about 80%, more preferably about 85%, 90%, 95% and 99% of all macromolecular species present in the composition. Optimally, the target species is purified to basic homogeneity (contaminant species cannot be detected in the composition by conventional detection methods), where the composition consists essentially of a single macromolecular species. In certain embodiments, the substantially pure material is at least 50% pure (ie, free of contaminants), more preferably at least 90% pure, more preferably at least 95% pure, still more preferably at least 98% pure, and most Jia is at least 99% pure.

As is known in the art, the term "identity" refers to the relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by comparing the sequences. In this technique, "identity" also means the degree of sequence relatedness between polypeptide or nucleic acid molecule sequences, as the case may be, as determined by the match between strings of nucleotide or amino acid sequences . "Consistency" measures the gap comparison by comparing the percentage of identical matches between two or more sequences addressed by a specific mathematical model of a computer program (that is, "algorithm").

The term "similarity" is a related concept, but compared to "identity", it refers to a similarity measure that includes consistent matching and conservative substitution matching. Since conservative substitutions apply to polypeptides rather than nucleic acid molecules, similarity only involves comparison of polypeptide sequences. If two polypeptide sequences, for example, 10 out of 20 amino acids are identical, and the remainder are all non-conservative substitutions, both the percent identity and similarity will be 50%. If there are 5 more positions with conservative substitutions in the same example, the percentage of identity is still 50%, but the percentage of similarity will be 75% (15/20). Therefore, in the presence of conservative substitutions, the degree of similarity between two polypeptide sequences will be higher than the percent identity between the two polypeptides.

A "fragment" or "portion" of a polypeptide or antibody according to the present invention can be prepared by truncation, for example, by removing one or more amino acids from the N and / or C-terminal ends of the polypeptide. Up to 10, up to 20, up to 30, up to 40 or more amino acids can be removed from the N and / or C terminal in this way. Fragments or parts can also be generated by one or more internal deletions.

The variant antibody may comprise 1, 2, 3, 4, 5, up to 10, up to 20, up to 30 or more amino acid substitutions and / or deletions and / or insertions of the specific sequences and fragments as described above. "Deleted" variants can include the deletion of individual amino acids; the deletion of smaller amino acid groups, such as 2, 3, 4, or 5 amino acids; or the deletion of larger amino acid regions, such as specific amines Loss of base acid domain or other characteristics. "Insertion" variants may include insertion of individual amino acids; insertion of smaller amino acid groups, such as 2, 3, 4, or 5 amino acids; or insertion of larger amino acid regions, such as insertion of specific amino acids Domain or other characteristics. "Substituted" variants preferably involve replacing one or more amino acids with the same number of amino acids and performing conservative amino acid substitutions. For example, the amino acid may be substituted with a replacement amino acid having similar characteristics, such as another basic amino acid, another acidic amino acid, another neutral amino acid, another charged amino acid, Another hydrophilic amino acid, another hydrophobic amino acid, another polar amino acid, another aromatic amino acid, or another aliphatic amino acid. Some characteristics of the 20 main amino acids that can be used to select suitable substituents are as follows:

The substitution variant removes at least one amino acid residue in the antibody molecule and inserts a different residue in its place. The most interesting sites for substitution-induced mutations include hypervariable regions, but they also cover structural changes. Conservative substitutions are shown in Table 1 entitled "Conservative substitutions". If such substitutions result in a change in biological activity, a more substantial change may be introduced named "exemplary substitution" as shown below or as further described below with regard to the amino acid type, and the product is screened.
Table 1
Amino acids and substitutions

Substantial modification of the biological characteristics of antibodies is achieved by selecting substitutions that maintain significantly different effects: (a) the structure of the polypeptide backbone in the substitution region, for example, in the form of beta sheets or helical configurations; Charge or hydrophobicity at the target; or (c) accumulation of side chains. Based on common side chain characteristics, naturally occurring residues are divided into the following groups:
i. Non-polar: leucine, Met, Ala, Val, Leu, Ile;
ii. Polarity, no charge: Cys, Ser, Thr, Asn, Gln;
iii. Acidic (negatively charged): ASP, Glu;
iv. Alkaline (positively charged): Lys, Arg;
v. Residues affecting chain orientation: Gly, Pro; and
vi. Aromatic: Trp, Tyr, Phe, His.

Non-conservative substitutions are made by exchanging one member of one of these categories for another category.

For example, one type of substitution that can be performed is to change one or more cysteine in a chemical reaction of the antibody to another residue such as (but not limited to) alanine or serine. For example, there may be substitution of atypical cysteine. Substitutions can be made in the CDR or framework regions or constant regions of antibody variable domains. In some embodiments, cysteine is typical. Any cysteine residues that are not involved in maintaining the proper configuration of the antibody can also generally be substituted with serine to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, especially when the antibody is an antibody fragment such as an Fv fragment, a cysteine bond can be added to the antibody to improve its stability.

"Antibodies" are immunoglobulin molecules that can specifically bind to targets such as carbohydrates, polynucleotides, lipids, and polypeptides via at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, this term covers not only complete multiple or monoclonal antibodies, but also unless specified, any antigen-binding fragments that compete with the intact antibody for specific binding, fusion proteins containing antigen-binding fragments, and antigen recognition sites Point to any other modified configuration of the immunoglobulin molecule. Antigen-binding fragments include, for example, Fab, Fab ', F (ab') ₂ , Fd, Fv, domain antibodies (dAb, such as shark and camel antibodies), fragments including complementarity determining regions (CDR), single-chain variable fragment antibodies ( scFv), maximal antibody, minibody, internal antibody, bifunctional antibody, trifunctional antibody, tetrafunctional antibody, v-NAR and biscFv and polypeptides containing at least fragments of immunoglobulins sufficient to bind specific antigens to the polypeptide. Antibodies include antibodies of any class, such as IgG, IgA, or IgM (or subclasses thereof), and the antibody need not be of any particular class. Immunoglobulins can be classified into different classes depending on the antibody amino acid sequence of the constant region of their heavy chains. There are five main types of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these immunoglobulins can be further divided into subclasses (isotypes), such as IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ , IgA ₁ and IgA ₂ . The heavy chain constant regions corresponding to different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunit structure and three-dimensional configuration of different classes of immunoglobulins are well known.

The term "antigen-binding portion" or "antigen-binding fragment" of an antibody (or only "antibody portion") as used interchangeably herein refers to one or more antibodies that retain specific binding to an antigen (e.g. CXCR5) Fragment. It has been shown that the antigen-binding function of antibodies can be performed by fragments of full-length antibodies. Examples of binding fragments covered by the term "antigen-binding fragment" of antibodies include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; (ii) F (ab ') 2 fragments, including A bivalent fragment of two Fab fragments connected by a disulfide bridge in the hinge region; (iii) Fd fragment composed of VH and CH1 domains; (iv) Fv fragment composed of VL and VH domains of one arm of an antibody ; (V) dAb fragment (Ward et al., (1989) Nature 341: 544-546), consisting of the VH domain; and (vi) isolated complementary determining region (CDR), disulfide-bonded Fv (dsFv) , And anti-idiotype (anti-Id) antibodies and internal antibodies. In addition, although the two domains VL and VH of the Fv fragment are encoded by separate genes, they can be joined using recombinant methods by making it possible to make synthetic linkers made in the form of a single protein chain, where the VL and VH regions are paired to form Monovalent molecules (referred to as single-chain Fv (scFv); see, for example, Bird et al. Science 242: 423-426 (1988) and Huston et al. Proc. Natl. Acad. Sci. USA 85: 5879-5883 (1988)). These single chain antibodies are also intended to be covered by the term "antigen-binding fragment" of an antibody. Other forms of single chain antibodies are also covered, such as bifunctional antibodies. Bifunctional antibodies are bivalent, bispecific antibodies in which the VH and VL domains appear on a single polypeptide chain, but use a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing this And other domains are paired with complementary domains of other chains and produce two antigen binding sites (see, for example, Holliger et al. Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993); Poljak et al., 1994, Structure 2: 1121-1123).

Antibodies can be derived from any mammal or other animal including (but not limited to) humans, monkeys, pigs, horses, rabbits, dogs, cats, mice, etc. such as birds (eg chickens), fish (eg sharks) and camels (eg camels) horse).

The "variable region" of an antibody refers to the variable region of the antibody light chain (VL) or the variable region of the antibody heavy chain (VH) alone or in combination. As is known in the art, the variable regions of the heavy and light chains are each composed of four framework regions (FR) connected by three "complementarity determining regions (CDR)" (also known as hypervariable regions (HVR)) And contribute to the formation of the antigen binding site of the antibody. If a variant of the variable region of the object is needed, especially in the case of replacing amino acid residues outside the CDR region (that is, in the framework region), it is suitable Amino acid substitutions, preferably conservative amino acid substitutions, can be identified by comparing the variable region of the subject with the variable regions of other antibodies whose variable regions contain the same typical category as the variable region of the subject CDR1 and CDR2 sequences (Chothia and Lesk, J. Mol. Biol. 196 (4): 901-917, 1987).

In certain embodiments, deterministic delineation of CDRs and confirmation of residues that include the binding site of an antibody are achieved by solving the structure of the antibody and / or solving the structure of the antibody-ligand complex. In some embodiments, it can be achieved by any of a variety of techniques known to those skilled in the art, such as X-ray crystallization. In some embodiments, various analytical methods can be used to identify or approximate identify CDR regions. In some embodiments, various analytical methods can be used to identify or approximate identify CDR regions. Examples of such methods include (but are not limited to) Kabat definition, Chothia definition, AbM definition contact definition and configuration definition.

Kabat is defined as the standard used to number residues in antibodies and is commonly used to identify CDR regions. See, for example, Johnson and Wu, 2000, Nucleic Acids Res., 28: 214-8. The Chothia definition is similar to the Kabat definition, but the Chothia definition considers the location of certain structural loop areas. See, for example, Chothia et al., 1986, J. Mol. Biol., 196: 901-17; Chothia et al., 1989, Nature, 342: 877-83. AbM defines an integrated package that uses a computer program that mimics antibody structures generated by Oxford Molecular Group. See, for example, Martin et al., 1989, Proc Natl Acad Sci (USA), 86: 9268-9272; "AbM ™, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd. AbM defines the use of a combination of a knowledge database and a complete algorithm to simulate the tertiary structure of antibodies from primary sequences, such as those described by: Samudrala et al., 1999, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach, "in PROTEINS, Structure, Function and Genetics Suppl., 3: 194-198.

The definition of Contact is based on the analysis of the available composite crystal structure. See, for example, MacCallum et al., 1996, J. Mol. Biol., 5: 732-45. In another approach (referred to herein as the "configuration definition" of the CDR), the position of the CDR can be identified as a residue that contributes to the enthalpy of antigen binding. See, for example, Makabe et al., 2008, Journal of Biological Chemistry, 283: 1156-1166. Although the definition of other CDR boundaries may not strictly follow one of the above methods, it will still overlap with at least a part of KabatCDR, but it can be shortened or extended according to the following predictions or experimental findings: specific residues or groups of residues do not significantly affect Antigen binding. As used herein, CDR can refer to a CDR defined by any method known in the art, including combinations of methods. The methods used herein may utilize CDRs defined according to any of these methods. For any given embodiment that contains more than one CDR, the CDR may be defined according to any one of Kabat definition, Chothia definition, extended definition, AbM definition, contact definition and / or configuration definition.

"Contact residues" as used herein with reference to antibodies or antigens specifically bound thereby refers to the amino acid residues present on the antibody / antigen, which contain at least one heavy atom (ie, non-hydrogen), in the presence of Within 4 Å or less of the heavy atoms of amino acid residues on homologous antibodies / antigens.

"Framework" (FR) residues are antibody variable domain residues other than CDR residues. The VH or VL domain framework includes four framework regions FR1, FR2, FR3, and FR4, interspersed with CDRs having the following structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

As stated previously herein, the residues in the variable domain are generally numbered according to Kabat, which is a numbering system for heavy chain variable domains or light chain variable domains used for antibody compilation. See Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th Edition. Public Health Service, National Institutes of Health, Bethesda, MD. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to the shortening or insertion of the FR or CDR of the variable domain. For example, the heavy chain variable domain may include a monoamino acid insertion after residue 52 of H2 (according to residue 52a of Kabat) and an embedded residue after residue FR 82 of the heavy chain (eg according to Kabat Residues 82a, 82b and 82c). For a given antibody, the Kabat numbering of residues can be determined by aligning the homologous region of the antibody sequence with the "standard" Kabat numbering sequence. Various algorithms for assigning Kabat numbers are available. The algorithm implemented in Abysis version 2.3.3 (www.abysis.org) can be used to assign Kabat numbers to variable regions CDR-L1, CDR-L2, CDR-L3, CDR-H2 and CDR-H3, and The AbM definition can then be used for CDR-H1.

As used herein, "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, that is, the individual antibodies that make up this population are the same except for mutations that may exist in small amounts and are likely to occur naturally. Monoclonal antibodies are highly specific for a single antigenic site. Furthermore, in contrast to multiple antibody preparations that usually include different antibodies directed against different determinants (antigenic determinants), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "single plant" indicates that the antibody is characterized by being obtained from a substantially homogeneous population of antibodies, and should not be understood as requiring the production of the antibody by any particular method. For example, the monoclonal antibodies used according to the present invention can be produced by the fusion tumor method first described by Kohler and Milstein, 1975, Nature 256: 495, or can be produced by methods such as US Pat. No. 4,816,567. Recombinant DNA method described. For example, monoclonal antibodies can also be isolated from phage libraries generated using the technique described in McCafferty et al., 1990, Nature 348: 552-554. As used herein, "humanized" anti-system refers to the following forms of non-human (eg, murine) antibodies: chimeric immunoglobulins, immunoglobulin chains, or fragments thereof containing minimal sequence derived from non-human immunoglobulin ( (Such as Fv, Fab, Fab ', F (ab') 2 or other antigen-binding sequences of antibodies). Preferably, the humanized antibody is a human immunoglobulin (receptor antibody) with the desired specificity, affinity, and ability, wherein the residues from the CDR of the receptor are derived from non-humans such as mice, rats, or rabbits Residue substitution of CDR of species (donor antibody). Humanized antibodies may contain the following residues: neither present in the recipient antibody nor in the imported CDR or framework sequences, but including such residues to further improve and optimize antibody performance.

The antibody or antigen-binding fragment of the present invention may be affinity matured. For example, affinity matured antibodies can be produced by procedures known in the art (Marks et al., 1992, Bio / Technology, 10: 779-783; Barbas et al., 1994, Proc Nat. Acad. Sci, USA 91 : 3809-3813; Schier et al., 1995, Gene, 169: 147-155; Yelton et al., 1995, J. Immunol., 155: 1994-2004; Jackson et al., 1995, J. Immunol., 154 (7 ): 3310-9; Hawkins et al., 1992, J. Mol. Biol., 226: 889-896; and WO2004 / 058184).

"Human antibodies" are those having the following amino acid sequences: corresponding to the amino acid sequences of antibodies produced by humans and / or manufactured using any of the techniques for making human antibodies disclosed herein. This definition of human antibody specifically excludes humanized antibodies that contain non-human antigen binding residues.

The term "chimeric antibody" is intended to mean that the variable region sequence is derived from one species and the constant region sequence is derived from an antibody of another species, such as the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody or vice versa Antibody. The term also encompasses antibodies that include a V region from one independent of one species (eg, first mouse) and a constant region from another independent of the same substance (eg, second mouse).

The term "antigen (Ag)" refers to a molecular entity used to immunize immunologically active vertebrates to produce antibodies (Abs) that recognize Ag or to screen performance libraries (such as phage, yeast, or ribosome display libraries and other libraries). In this article, Ag is more generally named and is generally intended to include target molecules specifically recognized by Ab, and therefore includes fragments or molecules used in the vaccination process for producing Ab or for selection of Ab for library screening. Mimicry. Therefore, for antibodies of the present invention that bind to CXCR5, the full-length CXCR5 from mammalian species (eg, human, monkey, mouse, and rat CXCR5) includes its monomers and multimers (such as dimers, trimers, etc.) Polymers, etc.) and truncated CXCR5 and other variants are called antigens.

In general, the term "antigenic determinant" refers to the region or region of the antigen (eg protein, nucleic acid, carbohydrate, etc.) to which the antibody specifically binds, that is, the region or region in contact with the antibody entity. Therefore, the term "antigenic determinant" refers to a portion of a molecule that can be recognized and bound by an antibody in one or more of its antigen-binding regions. Generally, epitopes are defined in the context of molecular interactions between "antibodies or their antigen-binding fragments" and their corresponding antigens. Epitopes are often composed of surface groupings of molecules such as amino acids or sugar side chains, and have special three-dimensional structural characteristics and special charge characteristics. In some embodiments, the epitope can be a protein epitope. Protein epitopes can be linear or conformal. In a linear epitope, all interaction points between a protein and an interacting molecule (such as an antibody) exist linearly along the first-order amino acid sequence of the protein. "Non-linear epitopes" or "configurational epitopes" are non-contiguous polypeptides (or amino acids) contained in antigen proteins that bind to antibodies specific for the epitopes. As used herein, the term "antigenic epitope" is determined by any method well known in the art (eg, by conventional immunoassay), which is defined as the part of the antigen that can specifically bind to the antibody . Alternatively, during the discovery process, the generation and characterization of antibodies can clarify information about the desired epitope. Based on this information, antibodies that bind to the same epitope can then be competitively screened. The way to achieve this is to perform competition and cross-competition studies to find antibodies that compete with each other or cross-compete for binding to CXCR5, such as antibodies that compete for binding to antigens.

"Priority binding" or "specific binding" (interchangeably used herein) antibodies to epitopes are terms that are clearly understood in the art, and the methods used to determine such specific or preferential binding are also It is well known in the art. A molecule is said to exhibit "specific binding" or "priority binding" if it reacts or associates with a particular cell or substance more frequently, faster, lasts longer, and / or has a greater affinity than an alternative cell or substance. ". If the antibody binds to the target with greater affinity, affinity, easier, and / or longer duration than the other substance, the antibody "specifically binds" or "prioritizes" to the target. In addition, if the antibody binds to the target in the sample with greater affinity, affinity, easier, and / or longer duration than it binds to other substances present in the sample, the antibody "specifically binds" or " "Priority binding" to the target. For example, an antibody that specifically or preferentially binds to a CXCR5 epitope has a greater affinity / binding power, is easier to bind to this epitope than to other CXCR5 epitopes or non-CXCR5 epitopes, and / or Antibodies that last longer. It should also be understood after reading this definition that, for example, an antibody (or portion or epitope) that specifically or preferentially binds to a first target may or may not be specific or preferentially binds to a second target. Thus, "specific binding" or "priority binding" does not necessarily require (though it may include) exclusive binding. Reference to bonding is general but does not necessarily mean preferential bonding. "Specific binding" or "preferential binding" includes compounds (such as proteins, nucleic acids, antibodies, and the like) that recognize and bind to specific molecules, but do not substantially recognize or bind other molecules in the sample. For example, an antibody or peptide receptor that recognizes and binds to a homologous ligand or binding partner in a sample (eg, an anti-CXCR5 antibody that binds CXCR5) but does not substantially recognize or bind to other molecules in the sample, specificity Binding to the homologous ligand or binding partner. Therefore, under specified analysis conditions, specific binding moieties (such as antibodies or antigen-binding fragments or receptors or ligand-binding fragments thereof) preferentially bind to specific target molecules and do not bind to other groups present in the test sample in large amounts Minute.

Various analysis formats can be used to select antibodies or peptides that specifically bind related molecules. For example, solid-phase ELISA immunoassay, immunoprecipitation, Biacore ™ (GE Healthcare, Piscataway, NJ), KinExA, fluorescent activated cell sorting (FACS), Octet ™ (FortéBio, Inc., Menlo Park, CA) and Western blot analysis and many other analytical methods can be used to identify antibodies that specifically react with antigens or receptors or ligand-binding fragments that specifically bind to homologous ligands or binding partners. Typically, the specific or selective reaction will be at least twice the background signal or noise, more usually more than 10 times the background, even more usually more than 50 times the background, more usually more than 100 times the background, more usually more than the background 500 times, even more often more than 1000 times the background and even more often more than 10,000 times the background. In addition, when the equilibrium dissociation constant (K _D ) ≤1 µM, preferably ≤100 nM, better ≤10 nm, even better ≤100 pM, more preferably ≤10 pM and even better ≤1 pM, it is said Antibodies "specifically bind" antigens. In some embodiments, when the equilibrium dissociation constant (K _D ) ≤ 7 nM, the antibody is said to "specifically bind" the antigen.

The term "binding affinity" is used herein as a measure of the strength of the non-covalent interaction between two molecules (and, for example, antibodies or fragments thereof and antigens). The term "binding affinity" is used to describe a monovalent interaction (intrinsic activity).

In addition, to determine the binding affinity of the CXCR5 antibody to CXCR5 expressing cells, a cell binding experiment can be performed to determine the apparent affinity. Antibody to cells expressing the target can be calculated as the apparent affinity equilibrium binding titration curves of EC _50, wherein the geometric mean fluorescence intensity of the population of antigen binding (GMFI) based quantified by flow cytometry measurement.

The binding affinity between two molecules (eg, an antibody or fragment thereof and an antigen) via a monovalent interaction can be quantified by measuring the dissociation constant (K _D ). Conversely, K _D can be determined by kinetic measurements of complex formation and dissociation using, for example, the surface plasmon resonance (SPR) method (Biacore). Association rate constant corresponding to the monovalent complex dissociation and association rate constant are referred to as k _a (or k _on) and dissociation rate constant k _d (or k _off). K _D via the equation _{K D = k d / k a} d k _a related and k. The dissociation constant value can be directly determined by well-known methods, and even the dissociation constant value of the composite mixture can be calculated by methods such as those described in Caceci et al. (1984, Byte 9: 340-362). For example, K _D can be established using a combination analysis such as the double filter nitrocellulose filter disclosed by Wong and Lohman (1993, Proc. Natl. Acad. Sci. USA 90: 5428-5432). Other standard analyses used to assess the binding capacity of ligands (such as antibodies against target antigens) are known in the art and include, for example, ELISA, Western blot, RIA, and flow cytometry analysis and Other analyses illustrated elsewhere in this article. The binding kinetics and binding affinity of antibodies can also be assessed by standard analysis known in the art, such as, for example, by surface plasmon resonance (SPR) using Biacore ™ system or KinExA.

A competitive binding analysis may be performed in which the antibody binding antigen is compared to another ligand to which the target binds, such as another antibody or soluble receptor that binds to the target in other ways. The concentration at which 50% inhibition occurs is called K _i . Under ideal conditions, K _i is equal to K _D. Because the value of K _i should never be less than K _D , it is advisable to replace the measurement of K _i by providing the upper limit of K _D.

Following the above definitions, may be related to the interaction of the comparison by the comparison of individual K antibody / antigen complex binding affinity value of _D (e.g. compare different antibodies on binding affinity of a given antigen) with different molecules. The K _D value of an antibody or other binding partner can be determined using the methods specified in this technique. One method of determining antibody K _D is by using surface plasmon resonance or using a biosensor system (such as the Biacore® system).

Similarly, the specific interaction may be measured, and by comparison of related interactions (e.g., specific interaction between the antibody and antigen) of K _D values associated with the non-interacting (e.g. binding is not known to the control CXCR5 antibodies ) To evaluate the K _D value.

An antibody which specifically binds to the target can bind its target with high affinity, which as discussed above exhibit a low K _D; and may bind with lower affinity to other non-target molecule. For example, the antibody may be at least 1 × 10 K _D ^{- 6} M or more, more preferably 1 × 10 ^{- 5} M or more, more preferably 1 × 10 ^{- 4} M or greater, more preferably 1 × 10 ^{- 3} M or more, even more preferably 1 × 10 ^- or more of the lower case ² M, bound to a non-target molecule. The antibody of the present invention is preferably capable of binding to its target, and its affinity is at least twice, 10 times, 50 times, 100 times, 200 times, 500 times, 1,000 times, or 10,000 Times or more.

The term "competition" as used herein in relation to an antibody means that the first antibody or antigen-binding fragment thereof binds to the epitope in a binding manner sufficiently similar to that of the second antibody or antigen-binding fragment thereof, so that the first antibody and its cognate antigen are determined The result of the binding of the base is detectably reduced in the presence of the second antibody compared to the binding of the first antibody in the absence of the second antibody. Alternatives may but need not be this: the binding of the second antibody to its epitope in the presence of the first antibody can also be reduced detectably. That is, the first antibody can inhibit the binding of the second antibody to its epitope, while the second antibody does not inhibit the binding of the first antibody to its respective epitope. However, when each antibody detectably inhibits the binding of another antibody to its cognate epitope or ligand to the same, greater or lesser extent, the antibodies are said to "cross-compete" with each other Their respective epitopes. The present invention covers both competitive and cross-competitive antibodies. Regardless of the mechanism by which this competition or cross-competition occurs (eg, steric hindrance, conformational changes, or binding to a common epitope or part thereof), skilled artisans will understand that such competition and / or Cross-competing antibodies are encompassed and applicable in the methods disclosed herein.

Standard competition analysis can be used to determine whether the two resistance systems compete with each other. A suitable analysis for antibody competition involves the use of Biacore technology, which can use surface plasmon resonance (SPR) technology, usually using a biosensor system (such as the BIACORE® system) to measure the degree of interaction. For example, SPR can be used in competitive binding inhibition assays in vitro to determine the ability of one antibody to inhibit the binding of a second antibody. Another analysis method for measuring antibody competition uses an ELISA-based method.

In addition, the high-throughput method of "grouping" based on antibody competition is described in International Patent Application No. WO 2003/48731. If one antibody (or fragment) reduces the binding of another antibody (or fragment) to CXCR5, there is competition. For example, sequential binding competition analysis can be used, where different antibodies are added sequentially. A first antibody can be added to achieve near saturation binding. Subsequently, a second antibody is added. If compared to a parallel analysis in the absence of the first antibody, no significant reduction in the binding of the second antibody to CXCR5 or the binding of the second antibody to CXCR5 is detected (eg, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90% reduction) (the value can be set to 100%) Compete for each other.

In addition, an exemplary antibody epitope grouping analysis is provided in Example 8, which uses domain exchange between human and mouse CXCR5 proteins to evaluate possible epitopes between several antibodies. The skilled artisan will understand from the teachings provided herein that there are various analyses known in the art that can be used to determine the binding of at least two types of targets relative to each other, and that such analyses are included herein.

CXCR5 antibodies can be characterized using methods well known in the art. For example, one method is to identify the epitope or "epitope localization" it binds. There are many methods known in the art for locating and characterizing the position of epitopes on proteins, including, for example, Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold The crystal structure, competition analysis, gene fragment performance analysis, and synthetic peptide-based analysis of the solubilized antibody-antigen complex described in Spring Harbor, New York, 1999. In an additional example, epitope localization can be used to determine the sequence that binds to the CXCR5 antibody. Epitope localization can be purchased from various sources, such as Pepscan Systems (Edelhertweg 15, 8219 PH Lelystad, The Netherlands) The epitope can be a linear epitope, that is, included in a single stretch of amino acids; or The conformational epitopes formed by the three-dimensional interaction of acids may not necessarily be included in a single stretch. Peptides of various lengths (eg, at least 4-6 amino acids long) can be isolated or synthesized (eg, in a recombinant manner) and used for binding analysis using CXCR5 antibodies.

In addition, epitopes that bind to CXCR5 antibodies can be determined in systemic screening by using overlapping peptides derived from CXCR5 sequences and measuring antibody binding. Based on gene fragment performance analysis, the open reading frame encoding CXCR5 can be randomly or by specific gene constructs divided into fragments and the reactivity of CXCR5 with the expression fragments of the antibody to be tested can be determined. Gene fragments can be generated, for example, by PCR, and then transcribed and translated into proteins in vitro in the presence of radioactive amino acids. The binding of the antibody to the radiolabeled CXCR5 fragment was then determined by immunoprecipitation and gel electrophoresis.

Certain epitopes can also be identified by using large libraries of random peptide sequences presented on the surface of phage particles (phage libraries) or yeasts (yeast display). Alternatively, the defined library of overlapping peptide fragments can be tested for binding to the test antibody in a simple binding analysis. In an additional example, antigen mutation induction, domain exchange experiments, and alanine scanning mutation induction can be performed to identify residues required, sufficient, and / or necessary for epitope binding. For example, mutant CXCR5 can be used to conduct alanine scanning mutation induction experiments, in which various residues of the CXCR5 polypeptide have been replaced with alanine. By assessing the binding of antibodies to mutant CXCR5, the importance of specific CXCR5 residues for antibody binding can be assessed.

Another method that can be used to characterize CXCR5 antibodies is to use competitive analysis using other antibodies that are known to bind to the same antigen (ie, various fragments on CXCR5) to determine whether the CXCR5 antibody system binds to the same epitope as other antibodies . Competition analysis is well known to those skilled in the art.

In addition, multiple experiments and calculations of epitope localization methods can be used to define and characterize the epitope of a given antibody / antigen binding pair at varying levels of detail. Experimental methods include mutation induction, X-ray crystallization, nuclear magnetic resonance (NMR) spectroscopy, hydrogen / deuterium exchange mass spectrometry (H / D-MS), and various competitive binding methods well known in the art. Since each method relies on a unique principle, the description of the epitope is closely related to the method used to determine it. Therefore, depending on the epitope localization method used, the epitope of a given antibody / antigen pair will be defined in different ways.

At its most detailed level, the epitope used for the interaction between Ag and Ab can be defined by the spatial coordinates of the atomic contacts present in the Ag-Ab interaction and their relative contribution to the binding thermodynamics Definition of information. To a lesser degree, the epitope can be characterized by defining the spatial coordinates of the atomic contact between Ag and Ab. To a less detailed degree, epitopes can be characterized by amino acid residues, which include, for example, by specific criteria (eg, by atoms in Ab and Ag (eg, heavy atoms, ie, non-hydrogen atoms) Distance) is defined. To a less detailed level, epitopes can be characterized by function, for example, by competitive binding with other Abs. The epitope can also be more generally defined as the included amino acid residues. For these amino acid residues, substitution with another amino acid will change the characteristics of the interaction between Ab and Ag ( (For example, using alanine scan).

According to the fact that the epitope localization method used depends on the fact that the description and definition of epitopes are obtained at different levels of detail, it is similarly possible to perform epitopes of different Abs on the same Ag at different levels of detail Compare.

If the epitope described on the amino acid level, for example, determined by the X-ray structure, contains the same amino acid residue group, it is said to be consistent. If the epitopes share at least one amino acid, these epitopes are said to overlap. If the epitopes do not share amino acid residues, these epitopes are said to be independent (unique).

If the binding of the corresponding antibodies is exclusive to each other, that is, the binding of one antibody excludes the simultaneous or continuous binding of the other antibody, then the epitope characterized by the competitive binding is called overlapping. If the antigen can accommodate the binding of two corresponding antibodies at the same time, the epitope is called independent (unique).

The definition of the term "paratope" is derived from the definition of "antigenic determinant" above from a reversed perspective. Therefore, the term "paratope" refers to the region or region on the antibody that specifically binds to the antigen, that is, the amino acid residue on the antibody that contacts the antigen (CXCR5) (as defined elsewhere in this document "contact").

The epitope and paratope of a given antibody / antigen pair can be identified by conventional methods. For example, the general location of the epitope can be determined by evaluating the antibody's ability to bind to different fragments or variant CXCR5 polypeptides. The specific amino acid (epitope) in CXCR5 in contact with the antibody and the specific amino acid (paratope) in the antibody in contact with CXCR5 can also use conventional methods such as those described in the examples to Determination. For example, antibodies and target molecules can be combined and the antibody / antigen complex can be crystallized. The crystal structure of the complex can be determined and used to identify specific sites of interaction between the antibody and its target.

The antibody according to the present invention can bind to the same epitope or domain of CXCR5 as the antibody of the present invention specifically disclosed herein. Analysis and assays that can be used for this identification include analysis to assess the competitive binding of the relevant antibody to the CXCR5 receptor to CXCR5, biological activity analysis as described in Examples 1 to 10, and analysis of antibody crystal structure.

The antibody of the present invention may have the ability to compete or cross-compete with another antibody of the present invention for binding to CXCR5 as described herein. For example, the antibodies of the present invention can compete with or cross-compete for binding to CXCR5 with the antibodies described herein, or to suitable fragments or variants of CXCR5 that bind to the antibodies disclosed herein.

That is, if the first antibody competes with the second antibody for binding to CXCR5, but when the second antibody first binds to CXCR5, the first antibody does not compete, it is considered to compete with the second antibody (also known as unidirectional competition). When an antibody competes with another antibody, whichever antibody binds to CXCR5 first, then the antibody cross-competes with another antibody to bind to CXCR5. Competing or cross-competing antibodies can be identified based on their ability to compete / cross-compete with known antibodies of the invention in standard binding assays. For example, competition / cross-competition can be indicated using, for example, SPR, ELISA analysis, or flow cytometry using the Biacore ™ system. Such competition / cross-competition may indicate that the two antibodies bind to identical, overlapping or similar epitopes.

The antibodies of the present invention can therefore be identified by a method that includes a binding analysis that assesses whether the test anti-system can compete / cross-compete with the reference antibody for binding sites on the target molecule. Methods for performing competitive binding analysis are disclosed herein and / or are well known in the art. For example, it may involve using the following conditions to bind the reference antibody of the invention to the target molecule: under these conditions, the antibody can bind to the target molecule. The antibody / target complex can then be exposed to the test / second antibody and the extent to which the test antibody can replace the reference antibody of the invention from the antibody / target complex can be assessed. Alternative methods may involve contacting the test antibody with the target molecule under conditions that allow antibody binding, and then adding the reference antibody of the invention capable of binding the target molecule and assessing that the reference antibody of the invention can replace the antibody / target complex The degree to which the test antibody of the substance may bind to the target (ie non-competing antibody) at the same time.

The ability of the test antibody to inhibit the binding of the reference antibody of the invention to the target proves that the test antibody can compete with the reference antibody of the invention for binding to the target and therefore the test antibody binds to the same or substantially the same CXCR5 as the reference antibody of the invention Epitopes or regions on proteins. Test antibodies identified in such methods as competing with the reference antibodies of the invention are also antibodies of the invention. The test antibody can bind to CXCR5 in the same region as the reference antibody of the present invention and can compete with the reference antibody of the present invention. This fact indicates that the test antibody can act as a ligand at the same binding site as the antibody of the present invention and test Antibodies can therefore mimic the effect of reference antibodies and therefore are antibodies of the invention. This can be confirmed by comparing the activity of CXCR5 in the presence of the test antibody to the activity of CXCR5 in the presence of the reference antibody using the analysis as described more fully herein and otherwise under the same conditions.

The reference antibody of the invention may be an antibody as described herein, such as 11G2, 41A10, 5H7, and any variant or portion thereof that retains the ability to bind to CXCR5 as described herein. The antibody of the present invention can bind to the same epitope as the reference antibody described herein or any variant or part thereof that retains the ability to bind to CXCR5 as described herein.

As previously described elsewhere herein, specific binding can be assessed with reference to the binding of antibodies to non-target molecules. This comparison can be made by comparing the ability of the antibody to bind to the target and to bind to another molecule. This comparison can be made in the assessment of K _D or K _i as described above. The other molecule used for such comparison may be any molecule that is not the target molecule. Preferably, the other molecule is not consistent with the target molecule. Preferably, the target molecule is not a fragment of the target molecule.

The other molecule used to determine specific binding may be independent of the target in terms of structure or function. For example, another molecule may be an unrelated substance or accompanying substance in the environment.

The other molecule used to determine specific binding may be another molecule that participates in the same in vivo pathway as the target molecule (ie, CXCR5). By ensuring that the antibody of the present invention is specific to CXCR5 compared to another molecule, undesirable cross-reactivity in vivo can be avoided.

The antibodies of the invention may retain the ability to bind to some molecules related to the target molecule.

Alternatively, the antibodies of the invention may have specificity for specific target molecules. For example, it may bind to one target molecule as described herein, but may not bind or may bind to a different target molecule as described herein with a significantly reduced affinity. For example, full-length mature human CXCR5 can be used as a target, but antibodies that bind to that target may not be able to bind to other CXCR5 proteins from other species, such as other mammalian CXCR5, for example, or may bind to it with less affinity. In some embodiments, the antibody binds to both human and mouse CXCR5.

An "Fc fusion" protein is a protein in which one or more polypeptides are operably linked to an Fc polypeptide. Fc fusion combines the Fc region of an immunoglobulin with a fusion partner.

The "native sequence Fc region" includes an amino acid sequence identical to the amino acid sequence of the Fc region present in nature. "Variant Fc region" includes amino acid sequences that are different from amino acid sequences of the native sequence Fc region by means of at least one amino acid modification, but retain at least one effector function of the native sequence Fc region. Preferably, the variant Fc region has at least one amino acid substitution compared to the native sequence Fc region or the Fc region of the parent polypeptide, for example, about one to about ten in the native sequence Fc region or the Fc region of the parent polypeptide Amino acid substitution, and more preferably about one to about five amino acid substitutions. In this context, the variant Fc region should preferably have at least about 80% sequence identity with the native sequence Fc region and / or with the Fc region parent polypeptide and optimally at least about 90% sequence identity with this, and better At least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% sequence identity with this.

As known in the art, the "constant region" of an antibody refers to the constant region of the antibody light chain or the constant region of the antibody heavy chain, alone or in combination.

As used interchangeably herein, the terms "IgG Fc region", "Fc region", "Fc domain" and "Fc" refer to a portion of an IgG molecule that is crystallizable with papain digestion of the IgG molecule Fragment related. As used herein, these terms refer to the antibody constant region excluding the first constant region immunoglobulin domain and further refer to the part of that region. Therefore, Fc refers to the last two constant region immunoglobulin domains of IgA, IgD and IgG, and the last three constant region immunoglobulin domains of IgE and IgM and the flexible hinge N-terminus of these domains, or section. For IgA and IgM, Fc may include the J chain. For IgG, Fc includes the immunoglobulin domains Cγ2 and Cγ3 (C gamma 2 and C gamma 3) and the hinge between Cγ1 (C gamma 1) and Cγ2 (C gamma 2). Although the boundaries of the Fc region can be changed, the human IgG heavy chain Fc region is usually defined to contain residues C226 or P230 relative to its carboxyl terminus, where the numbering is based on Edelman et al., 1969 as described by Kabat et al., 1991 , Proc. Natl. Acad. Sci. USA 63 (1): 78-85 EU index. Generally, the Fc domain contains about amino acid residues 236 to about 447 of the constant domain of human IgG1. An exemplary human wild-type IgG1 Fc domain amino acid sequence is set forth in SEQ ID NO: 31. The Fc polypeptide can refer to this independent region, or this region in the case of an antibody, or an antigen-binding fragment thereof, or an Fc fusion protein.

The heavy chain constant domain includes the Fc region and further includes the CH1 domain and hinge of the IgG heavy chain and the CH2 and CH3 (and optionally CH4 of IgA and IgE) domains.

The "functional Fc region" possesses at least one effector function of the native sequence Fc region. Exemplary "effector functions" include C1q binding; complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity; phagocytosis; down-regulation of cell surface receptors (eg, B cell receptors), etc. These effector functions generally require an Fc region to be combined with a binding domain (such as an antibody variable domain or antigen-binding fragment thereof) and can be evaluated using various analyses known in the art to assess the effector functions of these antibody.

"Native sequence Fc region" includes amino acid sequences that are identical to the amino acid sequences of the Fc region found in nature. Native sequence human Fc regions include native sequence human IgGl Fc regions (non-A and A isotypes); native sequence human IgG2 Fc regions; native sequence human IgG3 Fc regions; and native sequence human IgG4 Fc regions, and naturally occurring variants thereof.

"Variant Fc region" contains at least one amino acid modified amino acid sequence that differs from the amino acid sequence of the native sequence Fc region.

"Fc receptor" or "FcR" describes a receptor that binds to the Fc region of an antibody. In some embodiments, FcyR is a natural human FcR. In some embodiments, FcR is an FcR (gamma receptor) that binds an IgG antibody and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including dual gene variants and alternate splicing forms of these receptors. FcγRII receptors include FcγRIIA (“activated receptor”) and FcγRIIB (“inhibited receptor”), both of which have similar amino acid sequences that differ mainly in their cytoplasmic domain. The activated receptor FcyRIIA contains an immunoreceptor tyrosine-based activation motif (IT AM) in its cytoplasmic domain. The inhibitory receptor FcyRIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see Daeron, Annu. Rev. Immunol. 15: 203-234 (1997)). FcR is exemplified by Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991); Capel et al., Immunomethods 4: 25-34 (1994); and de Haas et al., J. Lab. Clin. Med. 126 : 330-41 (1995). Other FcRs, including those identified in the future, are covered by the term "FcR" herein.

The term "Fc receptor" or "FcR" also includes the neonatal receptor FcRn, which is responsible for transferring maternal IgG to the fetus (Guyer et al., J. Immunol. 117: 587 (1976) and Kim et al., J. Immunol. 24: 249 (1994)) and regulate the homeostasis of immunoglobulins. The measurement method of binding to FcRn is known (see, for example, Ghetie and Ward., Immunol. Today 18 (12): 592-598 (1997); Ghetie et al., Nature Biotechnology, 15 (7): 637-640 (1997); Hinton et al., J. Biol. Chem. 279 (8): 6213-6216 (2004); WO 2004/92219 (Hinton et al.)).

"Effective function" refers to the biological activity attributable to the Fc region of an antibody, which varies with the isotype of the antibody. Examples of antibody effector functions include: Clq binding and complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; cell surface receptors (eg B cell receptors) ) Downregulation; and B cell activation.

"Human effector cells" are white blood cells that express one or more FcRs and perform effector functions. In certain embodiments, the cells exhibit at least FcyRIII and perform ADCC effector functions. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, macrophages, cytotoxic T cells, and neutrophils. Effector cells can be isolated from natural sources, such as from blood.

"Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity that binds to Fc receptors present on certain cytotoxic cells (such as NK cells, neutrophils, and macrophages) The secreted Ig on (FcR) enables these cytotoxic effector cells to specifically bind to antigen-bearing target cells and then kill target cells with cytotoxins. The primary cells used to mediate ADCC, NK cells, express FcyRIII only, while mononuclear cells express FcyRI, FcyRII, and FcyRIII. The performance of FcR on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9: 457-92 (1991). To assess the ADCC activity of related molecules, in vitro ADCC analysis can be performed, such as the in vitro ADCC analysis described in US Patent No. 5,500,362 or 5,821,337 or US Patent No. 6,737,056 (Presta). The effector cells suitable for such analysis include PBMC and NK cells. Alternatively or additionally, the ADCC activity of the relevant molecule can be assessed in vivo, for example, in an animal model, such as the animal disclosed in Clynes et al. Proc. Natl. Acad. Sci. (USA) 95: 652-656 (1998) model. Additional antibodies with altered Fc region amino acid sequences and increased or decreased ADCC activity are described in, for example, US Patent No. 7,923,538 and US Patent No. 7,994,290.

An anti-system with "enhanced ADCC activity" refers to an antibody that is more effective than the parent antibody in regulating ADCC in vitro or in vivo, wherein the antibody and the parent antibody differ in at least one structural aspect, and when the When the amount of antibody and parent antibody used for analysis are substantially the same. In some embodiments, the antibody and the parent antibody have the same amino acid sequence, but the antibody is defucosylated and the parent antibody is fucosylated. In some embodiments, ADCC activity will be determined using in vitro ADCC analysis as disclosed herein, but covers other assays or methods for determining ADCC activity, such as animal models and the like. In some embodiments, antibodies with enhanced ADCC activity have enhanced affinity for FcyRIIIA. In some embodiments, antibodies with enhanced ADCC activity have enhanced affinity for FcyRIIIA (V158). In some embodiments, antibodies with enhanced ADCC activity have enhanced affinity for FcγRIIIA (F158).

Antibodies with "changed" FcR binding affinity or ADCC activity have increased or decreased FcR binding activity and / or ADCC activity compared to the parent antibody, where the antibody and parent antibody differ in at least one structure Appearance. An antibody that "displays" an "increased binding" to FcR binds at least one FcR with a higher affinity than the parent antibody. Antibodies that "display" and "reduced binding" to FcR bind at least one FcR with a lower affinity than the parent antibody. Compared to native sequence IgG Fc regions, these antibodies that show reduced binding to FcR may have little or no significant binding to FcR, for example, 0-20% binding to FcR.

"Enhanced affinity for Fcγ RIIIA" means that the antibody has a greater affinity for Fcγ RIIIA (also referred to as CD 16a in some examples) than the parent antibody, wherein the difference between the antibody and the parent antibody is at least one structural state kind. In some embodiments, the antibody and the parent antibody have the same amino acid sequence, but the antibody is defucosylated and the parent antibody is fucosylated. Any suitable method for determining the affinity for FcyRIIIA can be used. In some embodiments, the affinity for FcyRIIIA is determined by the methods described herein. In some embodiments, antibodies with enhanced affinity for FcyRIIIA have enhanced ADCC activity. In some embodiments, antibodies with enhanced affinity for FcyRIIIA have enhanced affinity for FcyRIIIA (V158). In some embodiments, an antibody with enhanced affinity for FcyRIIIA has enhanced affinity for FcyRIIIA (F158).

L-fucose is also called 6-deoxy-L-galactose, which is a monosaccharide, that is, some N-linked and O-linked glycans and glycolipid components in animals. See Becker and Lowe, Glycobiology 13: 41R-51R (2003). Fucose is usually added as a modification to glycans (including glycans linked to blood group antigens, selectins, and antibodies). Fucose can be attached to the glycan via the α (1,2) bond, α (1,3) bond, α (1,4) bond and α (1,6) bond of the specific fucosyltransferase. The α (1,2) -fucose bond is usually associated with the H blood group antigen. The α (1,3) -fucose and α (1,4) -fucose bonds are associated with the modification of LewisX antigen. The α (1,6) -fucose bond is associated with N-linked GlcNAc molecules (such as those on antibodies).

The carbohydrate portion of the present invention will be described with reference to commonly used nomenclature used to describe oligosaccharides. A review of carbohydrate chemistry using this nomenclature can be found in Hubbard and Ivatt (1981) Ann. Rev. Biochem. 50: 555-583. This nomenclature includes, for example, Man representing mannose; GIcNAc representing 2-N-acetylglucosamine; Gal representing galactose; Fuc representing fucose; and Glc representing glucose. Sialic acid is described by the abbreviated symbol NeuNAc, which means 5-N-acetyl neryl glycosyl acid; and NeuNGc means 5-hydroxy acetyl neryl glycosyl acid (IUB-IUPAC Joint Commission on Biochemical Nomenclature, 1982, J. Biol. Chem. 257: 3347-3351; (1982) J. Biol. Chem. 257: 3352).

The carbohydrate structure of the present invention appears on proteins expressed as N-linked oligosaccharides. "N-linked glycosylation" refers to the attachment of carbohydrate moieties to asparagine residues in the polypeptide chain via GlcNAc. N-linked carbohydrates all contain a common Man 1-6 (Man1-3) Manβ1-4GlcNAcβ1-4GlcNAcβ-R core structure. Therefore, in the described core structure, R represents the asparagine residue of the prepared glycoprotein. The sequence of the prepared protein will contain asparagine-X-serine, aspartame-X-threonine and aspartame-X-cysteine, of which X is proline acid ( Any amino acid other than Asn-Xaa-Ser / Thr). "O-linked" carbohydrates are in contrast characterized by a common core structure, which is a GalNAc attached to the hydroxyl group of threonine or serine but does not require a common sequence. Among N-linked carbohydrates, the most important is N-linked "complex" carbohydrates, such as the "double tentacle" structure described herein.

Skilled artisans will recognize that the glycoprotein immunoglobulin G (IgG) is associated with three types of composite biantennary structures that contain zero, one, or two halves commonly known as G0, G1, and G2, respectively. Lactose residues (Wormland et al., 1997, Biochemistry 36: 1370-1380). Regarding human antibody molecules of the IgG class, each has N-linked oligosaccharides attached to the amide side chain of Asn 297 at the β-4 corner of the inner surface of the CH2 domain of the Fc region (Beale and Feinstein, 1976, Q. Rev. Biophys. 9: 253-259; Jefferis et al., 1995, Immunol. Letts. 44: 111-117). The oligosaccharide part attached to Asn 297 of the IgG CH2 domain belongs to the complex two-antennary type, which has a recognized hexasaccharide core structure and different external sugar residues (see Jefferis et al., 1997, supra; Wyss and Wagner, 1996, Current Opinions in Biotech. 7: 409-416). The core structure (GIcNAc2Man3GIcNAc) is a typical feature of di-antennary oligosaccharides and is shown schematically in FIG. 1.

Because each core structure can have two equal parts of N-acetylglucosamine, core fucose and galactose or sialic acid external sugars, there are a total of 36 structurally unique oligosaccharides, which can occupy Asn 297 Points (Jefferis and Lund, see above). It will also be recognized that within a particular CH2 domain, the glycosylation at Asn 297 may be asymmetric due to the oligosaccharide chain attached to either Asn 297 residue within the two chain Fc domains different. For example, although heavy chains synthesized in cells secreting a single antibody may be homogenous in their amino acid sequences, they are generally differentially glycosylated, resulting in a large number of structurally unique Ig glycoforms.

The main type of complex oligosaccharide structure (also called "glycotype") found in the CH2 domain of IgG is depicted in International Patent Publication No. WO 99/22764, page 7.

According to the present invention, G0 refers to a two-antennary structure in which no terminal sialic acid (NeuAcs) is present; G1 refers to a two-antennary structure with one Gal and no NeuAcs and G2 refers to a two-antennary structure with two terminal Gal and no NeuAc Tentacle structure. See, for example, FIGS. 2A-2G, which depict exemplary structures of G0, G1, G-1, and G2.

"Defucosylated" antibodies or "fucose-deficient" anti-systems refer to IgGl or IgG3 isotype antibodies that lack fucose in their constant region glycosylation. When the core fucosylated biantennary complex oligosaccharide glycosylation is capped with up to 2 Gal residues, glycosylation of human IgG1 or IgG3 occurs at Asn297. In some embodiments, the defucosylated antibody lacks fucose at Asn297. Depending on the amount of terminal Gal residues, these structures are called G0, G1 (α 1, 6 or α 1, 3) or G2 glycan residues. See, for example, Raju, T. S., BioProcess Int. 1: 44-53 (2003). The CHO type glycosylation of antibody Fc is described in, for example, Routier, F. FL, Glycoconjugate J. 14: 201-207 (1997). Various antibody glycoforms are shown in Figures 2A-2G.

In some embodiments, a "fucosyl" or "defucosylated" (as used interchangeably herein) anti-system refers to an antibody that has been glycosyl engineered to lack core fucose. Antibodies with reduced fucose content in the glycan moiety have increased affinity for FcyRIIIa (CD16), and therefore have enhanced activity-dependent cytotoxicity (ADCC) activity. Fucosyl antibodies can be prepared using the Potelligent® CHOK1SV cell line (Lonza Biologics), which lacks the two alleles of the gene responsible for fucose addition (α1,6-fucosyltransferase). Fucosyl or reduced fucose antibodies can also be produced by modifying oligosaccharide biosynthetic activity in various ways. For example, the high control group's production in cell lines of the N - acetyl glucosamine - transferase III (GnTIII) and the second generation of overexpression of Fc constant region of an antibody of the points associated oligosaccharide structures and inhibits ROCK Fucosylated. In these expression systems, the level of GnTIII expression is related to the production of defucosylated IgG1 glycoforms and the resulting enhanced ADCC activity. Fucosylation in cell culture can also be reduced by using sugar analogs, such as but not limited to fucose analogs as described in WO 2012/019165. Therefore, defucosylated or fucose-reduced antibodies can be prepared using various methods well known in the art.

In some embodiments, the defucosylated antibody has enhanced affinity for FcyRIIIA. In some embodiments, the defucosylated antibody has enhanced affinity for FcyRIIIA (V158). In some embodiments, the defucosylated antibody has enhanced affinity for FcyRIIIA (F158).

"Glycotype" refers to the complex oligosaccharide structure, which contains bonds of various carbohydrate units. Such structures are described in, for example, Essentials of Glycobiology Varki et al., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1999), which also provides an overview of standard glycobiology nomenclature. Such glycoforms include but are not limited to G2, G1, G0, G-1, and G-2 (see, for example, International Patent Publication No. WO 99/22764).

"Glycosylation pattern" is defined as a pattern of carbohydrate units. These carbohydrate units are covalently attached to proteins (such as glycoforms) and sites at which glycoforms are covalently attached to proteins. , More specifically the peptide backbone of immunoglobulins.

In some embodiments, at least 85% of a batch of antibodies that are recombinantly expressed in CHO host cells without sugar modification are fucosylated at Asn297. When referring to a composition comprising a plurality of antibodies, if less than about 5% of the antibodies in the composition contain fucose at at least one Asn297, the antibody is defucosylated. Even better, the level of defucosylation is about 100%, that is, no fucose is detected on any of the heavy chain Asn297 glycoforms using standard methods for measuring fucosylation of antibodies . Methods for measuring fucose include any method known in the art, including the methods described herein. In some embodiments, fucose is not detectable in a composition comprising a plurality of defucosylated antibodies. In some embodiments, the defucosylated antibody has enhanced ADCC activity.

"Complement dependent cytotoxicity" or "CDC" refers to lysis of target cells in the presence of complement. The activation of the classical complement pathway is initiated by the first component of the complement system (Clq) binding to (of the appropriate subclass) antibodies that bind to their cognate antigens. To assess complement activation, CDC analysis can be performed, for example, as described in Gazzano-Santoro et al., J. Immunol. Methods 202: 163 (1996). Antibodies with varying amino acid sequences in the Fc region and increased or decreased Clq binding ability are described in, for example, US Patent No. 6,194,551 Bl, US Patent No. 7,923,538, US Patent No. 7,994,290, and WO 1999/51642. See also, for example, Idusogie et al., J.

As used herein, the terms "wild-type amino acid", "wild-type IgG", "wild-type antibody" or "wild-type mAb" refer to within a certain population (eg humans, mice, rats, cells, etc.) Naturally occurring amine or nucleic acid sequence.

The "CXC chemokine receptor type 5" or "CXCR5" used interchangeably herein is also referred to as "CD185" in this technology and the "Burkitt lymphoma receptor 1 (BLR1)" is G protein coupled receptors expressed on some cells. The term CXCR5 includes CXCR5 homologs and heterologs, including humans, cynomolgus monkeys, rats, rabbits and mice, among others. As used herein, "CXCR5" refers to mammalian CXCR5, such as human, rat or mouse and non-human primate, cattle, sheep or pig CXCR5. Non-limiting illustrative examples of CXCR5 include humans (see, for example, Gene Bank Deposit Number P60568, SEQ ID NO: 32), cynomolgus monkeys (see, for example, Gene Bank Deposit Number Q29615, SEQ ID NO: 33), and mice (SEQ ID NO: 34) CXCR5. The term "CXCR5" also covers fragments, variants, isoforms and other homologues of these CXCR5 molecules. The variant CXCR5 molecule is generally characterized by having the same type of activity as the naturally occurring CXCR5, such as the ability to bind the CXCR5 receptor, the ability to induce receptor-mediated activity, and the ability to bind or not bind to the antibodies or antigen-binding fragments of the invention Ability.

CXCR5 can contain one or more, two or more than two, three or more than three, four or more than four, five or more than five, six or more than six, seven or more Seven, eight or more than eight, nine or more than nine, ten or more than ten, twelve or more than twelve or fifteen or more than fifteen surfaces available Of CXCR5. Figure 6 shows the amino acid sequences of wild-type mouse and human CXCR5, with exemplary surface accessible residues underlined. When CXCR5 includes homomultimeric form of CXCR5, the target may include one or more, two or more than two, three or more than three, four or more than four, five or more than Five, six or more than six, seven or more than seven, eight or more than eight, nine or more than nine, ten or more than ten, twelve or more than ten Two or fifteen or more than fifteen surface available residues of the first subunit of CXCR5, and one or more, two or more than two, three or more than three, four One or more than four, five or more than five, six or more than six, seven or more than seven, eight or more than eight, nine or more than nine, ten or More than ten, twelve or more than twelve or fifteen or more than fifteen surface available residues of the second subunit of CXCR5. The target molecule may comprise known epitopes from CXCR5.

As previously stated, wild-type human CXCR5 (hCXCR5) and mice indicating (by underlining) various exemplary extracellular regions of the protein (labeled "N", "L1", "L2", and "3") The aligned amino acid sequence of CXCR5 (mCXCR5) is shown in FIG. 5.

As outlined elsewhere herein, certain positions of antibody molecules may vary. As used herein, "position" means the position of the protein in the sequence. The positions can be numbered sequentially or according to an established format. For example, the EU index and Kabat index can be used to number the amino acid residues of the antibody. For example, position 297 is the position in human antibody IgG1. As outlined above, the corresponding position is generally determined via alignment with other parent sequences.

As used herein, "residue" means a position in a protein and its related amino acid identity. For example, asparagine 297 (also known as Asn297, also known as N297) is a residue in the human antibody IgG1.

As used interchangeably in this article, the terms "Tfh cell" or "Tfh" or "bona fide Tfh" or "germination center Tfh cell" or "GC Tfh cell" refer to a job found in the germinal center (GC) Follicular helper T cells, which are structures composed of GC Tfh cells, GC B cells, follicular dendritic cells (FDC), macrophages, and stroma. See Crotty, 2014, Immunity 41 (4): 529-542. The Tfh cell line is recognized by the constitutive expression of the B-cell follicle homing receptor CXCR5. Functionally, Tfh cells provide guidance signals to B cells to guide homotypic transformation, somatic hypermutation and rapid cell division as seed germinal centers.

The terms "Tfh-like cells", "circulating Tfh cells" and "cTfh" used interchangeably herein refer to Tfh cells that have left the germinal center. After leaving the GC, the cells acquire a phenotype with less activation and less polarization and are called "circular follicular helper T cells" (cTfh) or cTfh) "Tfh-like cells". See Crotty, 2014, Immunity 41 (4): 529-542. These cells express CXCR5. Compared with germinal center Tfh cells (that is, "bona fide Tfh cells" or "GC Tfh cells" or "Tfh"), cTfh cells (that is, Tfh-like cells) exhibit reduced levels of ICOS, Bcl -6, And cell activation markers such as CD69 and HLA-DR, but maintaining the ability to stimulate Ab production and Ig class switching in B cells in vitro after reactivation with homologous antigens.

As known in the art, "polynucleotide" or "nucleic acid" as used interchangeably herein refers to a nucleotide chain of any length, and includes DNA and RNA. Nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or analogs thereof or any substrate that can be incorporated into the chain by DNA or RNA polymerase. The polynucleotide may include modified nucleotides, such as methylated nucleotides and the like. If present, modifications to the nucleotide structure can be imparted before or after the chain combination. The sequence of nucleotides may be interspersed with non-nucleotide components. The polynucleotide can be further modified after polymerization, such as by binding to the labeling component. Other types of modifications include, for example, "lid"; replacing one or more of the naturally occurring nucleotides with analogs; internucleotide modifications, such as those with uncharged bonds (eg, methyl phosphonate, phosphotriester , Phosphoramidates, carbamates, etc.) and modifications with charged bonds (eg phosphorothioate, phosphorodithioate, etc.), which contain proteins such as proteins (eg nucleases, toxins, antibodies, signal peptides) , Poly-L-lysine, etc.), they are modified with intercalating agents (such as acridine, psoralen, etc.), they contain chelating agents (such as metals, radioactive metals, boron, Modification of metal oxides, etc., their modification containing alkylating agents, their modification with modified bonds (for example, alpha-mutated nucleic acids, etc.), and unmodified forms of polynucleotides. In addition, any hydroxyl groups generally present in sugars can be replaced, for example, by phosphonate groups, phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages with additional nucleotides, or can be bonded to Solid support. The 5 'and 3' terminal OH can be phosphorylated or substituted with an amine or organic end-capping group of 1 to 20 carbon atoms. Other hydroxyl groups can also be derived as standard protecting groups. Polynucleotides may also contain similar forms of ribose or deoxyribose generally known in the art, including, for example, 2'-O-methyl-, 2'-O-allyl, 2'-fluoro- or 2 '-Azido-ribose, carbocyclic sugar analogs, α- or β-mutated isomers, epimers (such as arabinose, xylose or lyxose); piperanose, furanose, Rhodiola heptulose, acyclic analogs and abasic nucleoside analogs (such as methyl riboside). One or more phosphodiester bonds can be replaced by alternative linking groups. These alternative linking groups include, but are not limited to, the following embodiments: wherein the phosphate ester is P (O) S ("thiothioate"), P (S) S ("dithioester"), ( O) NR ₂ ("Amidyl Ester"), P (O) R, P (O) OR ', CO or CH ₂ ("Methylal"), where each R or R' is independently H or visual In the case of a substituted or unsubstituted alkyl group (1-20 C) containing an ether (-O-) bond, aryl group, alkenyl group, cycloalkyl group, cycloalkenyl group or arylaldehyde group. Not all bonds in a polynucleotide need to be consistent. The foregoing description applies to all polynucleotides mentioned herein, including RNA and DNA.

As used herein, "vector" means a construct that is capable of delivering and preferably expressing one or more related genes or sequences in a host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plastids, slime or phage vectors, DNA or RNA expression vectors combined with cationic condensing agents, DNA encapsulated in liposomes or RNA expression vectors, and certain eukaryotic cells, such as producer cells.

"Host cells" include individual cells or cell cultures that can be or have become recipients of vectors for incorporation of polynucleotide inserts. Host cells include the progeny of a single host cell, and the progeny may be due to natural, accidental, or intentional mutations and may not necessarily be completely identical to the original mother cell (in terms of morphology or genomic DNA complement). Host cells include cells transfected and / or transformed in vivo with the polynucleotide of the present invention.

The host cell may be a prokaryotic cell or a eukaryotic cell. Exemplary eukaryotic cells include mammalian cells, such as primate or non-primate cells; fungal cells, such as yeast; plant cells; and insect cells.

Any host cell sensitive to cell culture and sensitive to protein or polypeptide expression can be used in accordance with the present invention. In certain embodiments, the host cell line is a mammal. Mammalian cell lines that can be used as hosts for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC). Non-limiting exemplary mammalian cells include but are not limited to NS0 cells, HEK 293 and Chinese hamster ovary (CHO) cells, and derivatives thereof, such as 293-6E and CHO DG44 cells, CHO DXB11, and Potelligent® CHOK1SV cells (BioWa / Lonza, Allendale, NJ). Mammalian host cells also include, but are not limited to, human cervical cancer cells (HeLa, ATCC CCL 2), infant hamster kidney (BHK, ATCC CCL 10) cells, monkey kidney cells (COS), and human hepatocyte cancer cells (such as Hep G2 ). Other non-limiting examples of mammalian cells that can be used according to the present invention include human retinoblastoma (PER.C6®; CruCell, Leiden, The Netherlands); monkey kidney CV1 transformed with SV40 (COS-7, ATCC CRL 1651) Cell lines; human embryonic kidney cell line 293 (HEK 293) or 293 cells (Graham et al., 1977, J. Gen Virol. 36:59) subcultured for growth in suspension culture; mouse plug Trieth cells (TM4, Mather, 1980, Biol. Reprod. 23: 243-251); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); dogs Kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse breast tumor cells (MMT 060562, ATCC CCL51); TR1 cells (Mather et al., 1982, Annals NY Acad. Sci. 383: 44-68); MRC 5 cells; FS4 cells; human liver cancer cell line (Hep G2); and a large number of myeloma Cell lines, including but not limited to BALB / c mouse myeloma cell line (NS0 / 1, ECACC number: 85110503), NS0 cells and Sp2 / 0 cells.

In addition, any number of commercially available and non-commercially available cell lines expressing polypeptides or proteins can be used according to the present invention. Those skilled in the art will understand that different cell lines may have different nutritional requirements and / or may require different culture conditions to achieve optimal growth and polypeptide or protein performance, and will be able to modify the conditions as needed.

The present invention includes any eukaryotic expression system known in the art or disclosed herein for producing related proteins, such as expression in insect cell systems, yeast expression systems, or mammalian cell systems, such as but not limited to CHO cell.

As used herein, "expression control sequence" means a nucleic acid sequence that directs transcription of a nucleic acid. The expression control sequence may be a promoter, such as a constitutive or inducible promoter; or an enhancer. The expression control sequence is operably linked to the nucleic acid sequence to be transcribed.

As used herein, "treatment" is a method used to obtain beneficial or desired clinical results. For the purposes of the present invention, beneficial or desired clinical outcomes include (but are not limited to) one or more of the following: increased survival (reduced mortality), decreased inflammation of the disease, decreased tissue fibrosis, disease pathology The appearance is improved, the pathological lesion is limited to local sites, the degree of damage from the disease is reduced, the duration of the disease is reduced and / or the number, degree or duration of symptoms related to the disease is reduced. The term includes administration of a compound or agent of the present invention to prevent or delay the onset of symptoms, complications or biochemical indicators of the disease, alleviate such symptoms or retard or inhibit the further development of the disease, condition or disorder. Treatment can be preventive (to prevent or delay the onset of the disease or prevent its clinical or subclinical symptoms from appearing) or therapeutically suppress or alleviate the symptoms after the disease appears.

"Improvement" means that one or more symptoms are alleviated or improved compared to the failure to administer the CXCR5 antibody. "Improvement" also includes shortening or reducing the duration of symptoms.

As used herein, an "effective dosage form" or "effective amount" of a drug, compound, or pharmaceutical composition is an amount sufficient to affect any of one or more beneficial or desired results. In a more specific aspect, an effective amount prevents, alleviates or ameliorates the symptoms of disease or infection and / or prolongs the survival of the treated individual. For prophylactic use, beneficial or desired results include elimination or reduction of the risk of the disease, reduction of the severity of the disease, or delay of the onset of the disease, where the disease includes the disease, its complications, and organisms exhibiting intermediate pathological phenotypes during the development of the disease Chemical, tissue and / or behavioral symptoms. For therapeutic use, beneficial or desired results include such as reducing symptoms of one or more of CXCR5-mediated diseases, disorders or conditions, reducing the dose of other drugs required to treat the disease, enhancing the effects of another drug and / or delaying The clinical outcome of the patient's disease progression. The effective dose can be administered in one or more forms of administration. For the purposes of the present invention, the effective dose of a drug, compound or pharmaceutical composition is an amount sufficient to directly or indirectly achieve prophylactic or therapeutic treatment. As understood in clinical situations, an effective dose of a drug, compound, or pharmaceutical composition may or may not be achieved together with another drug, compound, or pharmaceutical composition. Therefore, an "effective dose" can be considered as one or more therapeutic agents administered, and if the same or more other agents can be achieved or a desired result can be achieved, a single agent can be considered to be given in an effective amount.

"Individual" or "subject" is a mammal, more preferably a human. Mammals also include (but are not limited to) agricultural animals (eg cows, pigs, horses, chickens, etc.), sport animals, pets, primates, horses, dogs, cats, mice and rats. In some embodiments, the individual is deemed to be at risk for a disease, disorder, or condition that is mediated or associated with CXCR5 binding to its receptor and signaling thereby mediated. In certain embodiments, the individual has an autoimmune disease, disorder, or condition, such as type 1 diabetes. In certain embodiments, the human individual requires immunosuppressive therapy.

As used herein, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any material that, when combined with an active ingredient, allows the ingredient to retain biological activity and does not react with the individual's immune system. Examples include, but are not limited to, any of the carriers in standard medicine, such as phosphate buffered saline solutions, water, emulsions (such as oil / water emulsions), and various types of wetting agents. The preferred diluent for aerosol or parenteral administration is phosphate buffered saline (PBS) or physiological saline (0.9%). Compositions containing these carriers are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th Edition, A. Gennaro, Ed., Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy 20th edition, Mack Publishing, 2000).

Exemplary methods and materials are described herein, but methods and materials similar or equivalent to those described herein can also be used to practice or test the invention. These materials, methods, and examples are illustrative only and are not intended to be limiting.
II. Anti-CXCR5 antibody

The present invention relates to antibodies that bind to CXCR5. Preferably, the antibody specifically binds to CXCR5, that is, it binds to CXCR5 but it does not detectably bind to other molecules, or binds to other molecules with lower affinity. The invention further relates to anti-CXCR5 antibodies that exhibit altered effector functions. In some embodiments, the changing effect function is increased ADCC. In some embodiments, the antibody lacks or contains detectably reduced levels of fucose (ie, it is defucosylated). The present invention also relates to compositions containing such antibodies; and uses of such antibodies, including therapeutic and medical uses.

In one embodiment, the invention provides any of the following, or a composition (including a pharmaceutical composition) comprising: a light chain sequence or a fragment thereof, and a heavy chain or a fragment thereof, derived from the following antibodies Antibody of any one: 11G2, 41A10 and 5H7.

Antibodies suitable for the present invention may include monoclonal antibodies, multiple antibodies, antibody fragments (such as Fab, Fab ', F (ab') ₂ , Fv, Fc, etc.), chimeric antibodies, bispecific antibodies, hetero-binding antibodies , Single chain (scFv), mutants thereof, fusion proteins containing antibody fragments (such as domain antibodies), humanized antibodies, and any other modified configurations of immunoglobulin molecules that contain antigens of the desired specificity Recognition sites (including glycosylation variants of antibodies), amino acid sequence variants of antibodies, and covalently modified antibodies. The antibody may be mouse, rat, human, or any other source (including chimeric or humanized antibodies). In some embodiments, the CXCR5 anti-system monoclonal antibody. In some embodiments, the anti-systemic human or humanized antibody.

The CXCR5 antibody of the present invention can be produced by any method known in the art. General techniques for producing human and mouse antibodies are known in the art and / or described herein.

After initial identification, the activity of the candidate CXCR5 antibody can be further confirmed and refined by biological analysis known to test the targeted biological activity. In some embodiments, in vitro cell analysis is used to further characterize candidate CXCR5 antibodies. For example, bioanalysis can be used to screen candidates directly. The examples describe in detail some methods for identifying and characterizing CXCR5 antibodies.

In some aspects, the antibody or antigen-binding fragment thereof contains at least one amino acid sequence selected from the group consisting of mouse 11G2 VH, mouse 11G2 VL, h11G2 VH (XC155), h11G2 VH ( XC156), h11G2 VH (XC157), h11G2 VH (XC350), h11G2 VH (XC351), h11G2 VH (XC352), h11G2 VH (XC353), h11G2 VH (XC354), h11G2 VL (XC151), h11G2 VL (X153 , H11G2 VL (XC154), h11G2 VL (XC346), h11G2 VL (XC347), h11G2 VL (XC348), h11G2 VL (XC349), mouse 41A10 VH, mouse 41A10 VL, h41A10 VH (XC147), h41A10 VH ( XC148), h41A10 VH (XC150), h41A10 VL (XC142), h41A10 VL (XC143), h41A10 VL (XC144), h41A10 VL (XC145), h41A10 VL (XC146), h41A10 VL (XC149), mouse 5H7 VH and Mouse 5H7 VL.

In some aspects, the antibody or antigen-binding fragment competes with any of the above antibodies for binding to CXCR5.

In some aspects, the antibody or antigen-binding fragment thereof comprises a VH amino acid sequence and a VL amino acid sequence, which comprises the following combination: an antibody or antigen that competes with one or more of the following for binding to human CXCR5 Binding fragments: mouse 11G2, chimeric 11G2, h11G2 VH (XC152) / VL (XC151), h11G2 VH (XC152) / VL (XC153), h11G2 VH (XC152) / VL (XC154), h11G2 VH (XC152) / VL (XC346), h11G2 VH (XC152) / VL (XC347), h11G2 VH (XC152) / VL (XC348), h11G2 VH (XC152) / VL (XC349), h11G2 VH (XC155) / VL (XC151), h11G2 VH (XC155) / VL (XC153), h11G2 VH (XC155) / VL (XC154), h11G2 VH (XC155) / VL (XC346), h11G2 VH (XC155) / VL (XC347), h11G2 VH (XC155) / VL (XC3484), h11G2 VH (XC155) / VL (XC349), h11G2 VH (XC156) / VL (XC151), h11G2 VH (XC156) / VL (XC153), h11G2 VH (XC156) / VL (XC154), h11G2 VH (XC156) / VL (XC346), h11G2 VH (XC156) / VL (XC347), h11G2 VH (XC156) / VL (XC348), h11G2 VH (XC156) / VL (XC349), h11G2 VH (XC157) / VL ( XC151), h11G2 VH (XC157) / VL (XC153), h11G2 VH (XC157) / VL (XC154), h11G2 VH (XC157) / VL (XC346), h11G2 VH (XC157) / VL (XC347), h11G2 VH ( XC157) / VL (XC348), h11G2 VH (XC157) / VL (XC349), h11G2 VH (XC350) / VL (XC151), h11G2 VH (XC350) / VL (XC153), h11G2 VH (XC350) / VL (XC154) ), H11G2 VH (XC350) / VL (XC346), h11G2 VH (XC350) / VL (XC347), h11G2 VH (XC350) / VL (XC348), h11G2 VH (XC350) / VL (XC349), h11G2 VH (XC351) / VL ( XC151), h11G2 VH (XC351) / VL (XC153), h11G2 VH (XC351) / VL (XC154), h11G2 VH (XC351) / VL (XC346), h11G2 VH (XC351) / VL (XC347), h11G2 VH ( XC351) / VL (XC348), and h11G2 VH (XC351) / VL (XC349), h11G2 VH (XC352) / VL (XC151), h11G2 VH (XC352) / VL (XC153), h11G2 VH (XC352) / VL ( XC154), h11G2 VH (XC352) / VL (XC346), h11G2 VH (XC352) / VL (XC347), h11G2 VH (XC352) / VL (XC348), h11G2 VH (XC352) / VL (XC349), h11G2 VH ( XC353) / VL (XC151), h11G2 VH (XC353) / VL (XC153), h11G2 VH (XC353) / VL (XC154), h11G2 VH (XC353) / VL (XC346), h11G2 VH (XC353) / VL (XC347) ), H11G2 VH (XC353) / VL (XC348), h11G2 VH (XC353) / VL (XC349), h11G2 VH (XC354) / VL (XC151), h11G2 VH (XC354) / VL (XC153), h11G2 VH (XC354) ) / VL (XC154), h11G2 VH (XC354) / VL (XC346), h11G2 VH (XC354) / VL (XC347), h11G2 VH (XC354) / VL (XC348), h11G2 VH (XC354) / VL (XC349) , Mouse 41A10, chimeric 41A10, h41A10 VH (XC147) / VL (XC142), h41A10 VH (XC147) / VL (XC143), h41 A10 VH (XC147) / VL (XC144), h41A10 VH (XC147) / VL (XC145), h41A10 VH (XC147) / VL (XC146), h41A10 VH (XC147) / VL (XC149), h41A10 VH (XC148) / VL (XC142), h41A10 VH (XC148) / VL (XC143), h41A10 VH (XC148) / VL (XC144), h41A10 VH (XC148) / VL (XC145), h41A10 VH (XC148) / VL (XC146), h41A10 VH (XC148) / VL (XC149), h41A10 VH (XC150) / VL (XC142), h41A10 VH (XC150) / VL (XC143), h41A10 VH (XC150) / VL (XC144), h41A10 VH (XC150) / VL (XC145), h41A10 VH (XC150) / VL (XC146), h41A10 VH (XC150) / VL (XC149), mouse 5H7, and chimeric 5H7.

In some aspects, the antibody or antigen-binding fragment competes with any of the above antibodies for binding to CXCR5.

In some aspects, the antibody or antigen-binding fragment thereof comprises SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 35, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, CDR-L1, CDR-L2, and CDR-L3 described in the amino acid sequence of at least one of SEQ ID NO: 50 and SEQ ID NO: 51.

In some aspects, the antibody or antigen-binding fragment thereof further comprises SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 36, SEQ ID NO: 52, SEQ ID NO: 53 , CDR-H1, CDR-H2, and CDR-H3 described in the amino acid sequence of at least one of SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57;

In some aspects, the antibody or antigen-binding fragment thereof comprises CDR-L1, CDR-L2, CDR-L3 as set forth in the amino acid sequence SEQ ID NO: 1, and as the amino acid sequence SEQ ID NO: 6 CDR-H1, CDR-H2, and CDR-H3 described in

In some aspects, the antibody or antigen-binding fragment thereof comprises CDR-L1, CDR-L2, CDR-L3 as set forth in the amino acid sequence SEQ ID NO: 1, and as the amino acid sequence SEQ ID NO: 10 CDR-H1, CDR-H2, and CDR-H3 described in

In some aspects, the antibody or antigen-binding fragment thereof comprises CDR-L1, CDR-L2, CDR-L3 as set forth in the amino acid sequence SEQ ID NO: 1, and as the amino acid sequence SEQ ID NO: 12 CDR-H1, CDR-H2, and CDR-H3 described in

In some aspects, the antibody or antigen-binding fragment thereof comprises CDR-L1, CDR-L2, CDR-L3 as set forth in the amino acid sequence SEQ ID NO: 1, and as the amino acid sequence SEQ ID NO: 52 CDR-H1, CDR-H2, and CDR-H3 described in

In some aspects, the antibody or antigen-binding fragment thereof further comprises CDR-H1, CDR-H2 and CDR-H3 as set forth in the amino acid sequence SEQ ID NO: 6, and as SEQ ID NO: 1, SEQ ID NO: 5, in the amino acid sequence of at least one of SEQ ID NO: 35, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 51 The CDR-L1, CDR-L2 and CDR-L3 described.

In some aspects, the antibody or antigen-binding fragment thereof further comprises CDR-H1, CDR-H2, and CDR-H3 as set forth in the amino acid sequence SEQ ID NO: 10, and as SEQ ID NO: 1, SEQ ID NO: 5, in the amino acid sequence of at least one of SEQ ID NO: 35, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 51 The CDR-L1, CDR-L2 and CDR-L3 described.

In some aspects, the antibody or antigen-binding fragment thereof further comprises CDR-H1, CDR-H2, and CDR-H3 as set forth in the amino acid sequence SEQ ID NO: 12, and as SEQ ID NO: 1, SEQ ID NO: 5, in the amino acid sequence of at least one of SEQ ID NO: 35, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 51 The CDR-L1, CDR-L2 and CDR-L3 described.

In some aspects, the antibody or antigen-binding fragment thereof further comprises CDR-H1, CDR-H2, and CDR-H3 as set forth in the amino acid sequence SEQ ID NO: 52, and as SEQ ID NO: 1, SEQ ID NO: 5, in the amino acid sequence of at least one of SEQ ID NO: 35, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 and SEQ ID NO: 51 The CDR-L1, CDR-L2 and CDR-L3 described.

In some aspects, the antibody or antigen-binding fragment thereof further comprises CDR-H1, CDR-H2, and CDR-H3 as set forth in the amino acid sequence SEQ ID NO: 27, and as the amino acid sequence SEQ ID NO: CDR-L1, CDR-L2 and CDR-L3 described in 26.

In some aspects, the antibody or antigen-binding fragment thereof comprises CDR-L1, CDR-L2, and CDR as described in the amino acid sequence encoded by the insert with the plastid with accession number PTA-124324 deposited with ATCC -L3.

In some aspects, the antibody or antigen-binding fragment thereof comprises CDR-H1, CDR-H2, and CDR as described in the amino acid sequence encoded by the insert with the plastid with accession number PTA-124323 deposited with ATCC. -H3.

In some aspects, the antibody or antigen-binding fragment thereof comprises CDR-L1, CDR-L2, and CDR-L3 amino acid sequences encoded by an insert deposited with ATCC and having a plastid with accession number PTA-124324 Deposited in the ATCC, the amino acid sequence CDR-H1, CDR-H2 and CDR-H3 amino acid sequence encoded by the plastid with accession number PTA-124323.

In some aspects, the antibody or antigen-binding fragment competes with any of the above antibodies for binding to CXCR5.

In some aspects, the antibody or antigen-binding fragment thereof comprises: CDR-L1 comprising the amino acid sequence SEQ ID NO: 2, CDR-L2 comprising the amino acid sequence SEQ ID NO: 3, and SEQ-L2 comprising the amino acid sequence SEQ ID NO: 4 CDR-L3, amino acid sequence SEQ ID NO: 7 CDR-H1, amino acid sequence SEQ ID NO: 8 CDR-H2, and amino acid sequence SEQ ID NO: 9 CDR-H3.

In some aspects, the antibody or antigen-binding fragment thereof comprises: CDR-L1 comprising the amino acid sequence SEQ ID NO: 2, CDR-L2 comprising the amino acid sequence SEQ ID NO: 3, and SEQ-L2 comprising the amino acid sequence SEQ ID NO: CDR-L3 of 4, CDR-H1 of amino acid sequence SEQ ID NO: 7, CDR-H2 of amino acid sequence SEQ ID NO: 8, and CDR-H2 of amino acid sequence SEQ ID NO: 11 CDR-H3.

In some aspects, the antibody or antigen-binding fragment thereof comprises: CDR-L1 comprising the amino acid sequence SEQ ID NO: 2, CDR-L2 comprising the amino acid sequence SEQ ID NO: 3, and SEQ-L2 comprising the amino acid sequence SEQ ID NO: 4 CDR-L3, amino acid sequence SEQ ID NO: 19 CDR-H1, amino acid sequence SEQ ID NO: 20 CDR-H2, and amino acid sequence SEQ ID NO: 21 CDR-H3.

In some aspects, the antibody or antigen-binding fragment thereof comprises: the CDR-L1 comprising the amino acid sequence SEQ ID NO: 14, the CDR-L2 comprising the amino acid sequence SEQ ID NO: 15, the CDR-L2 comprising the amino acid sequence SEQ ID NO: CDR-L3 of 16, CDR-H1 of amino acid sequence SEQ ID NO: 19, CDR-H2 of amino acid sequence SEQ ID NO: 20, and SEQ ID NO: 21 of amino acid sequence CDR-H3.

In some aspects, the antibody or antigen-binding fragment thereof comprises: the CDR-L1 comprising the amino acid sequence SEQ ID NO: 14, the CDR-L2 comprising the amino acid sequence SEQ ID NO: 15, the CDR-L2 comprising the amino acid sequence SEQ CDR-L3 of ID NO: 16, CDR-H1 of amino acid sequence SEQ ID NO: 7, CDR-H2 of amino acid sequence SEQ ID NO: 8, and CDR-H2 of amino acid sequence SEQ ID NO: 9 CDR-H3.

In some aspects, the antibody or antigen-binding fragment thereof comprises: the CDR-L1 comprising the amino acid sequence SEQ ID NO: 14, the CDR-L2 comprising the amino acid sequence SEQ ID NO: 15, the CDR-L2 comprising the amino acid sequence SEQ CDR-L3 of ID NO: 16, CDR-H1 of amino acid sequence SEQ ID NO: 8, CDR-H2 of amino acid sequence SEQ ID NO: 9, and CDR-H2 of amino acid sequence SEQ ID NO: 10 CDR-H3.

In some aspects, the antibody or antigen-binding fragment thereof comprises the sequence SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 36, SEQ ID NO: 52, SEQ ID NO: 53 , CDR-H1, CDR-H2, and CDR-H3 amino acid sequences described in at least one of SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, and SEQ ID NO: 57.

In some aspects, the antibody or antigen-binding fragment thereof comprises the sequence SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 35, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49 , CDR-L1, CDR-L2, and CDR-L3 amino acid sequences set forth in at least one of SEQ ID 50 and SEQ ID NO 51.

In some aspects, the antibody or antigen-binding fragment thereof comprises the CDR-L1, CDR-L2, and CDR-L3 amino acid sequences as set forth in SEQ ID NO: 5.

The antibody or antigen-binding fragment thereof may comprise a VH comprising an amino acid sequence that is at least 90% identical to the amino acid sequence SEQ ID NO: 6. The VH may comprise an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence SEQ ID NO: 6. VH may comprise the amino acid sequence SEQ ID NO: 6.

The antibody or antigen-binding fragment thereof may comprise a VH comprising an amino acid sequence at least 90% identical to the amino acid sequence SEQ ID NO: 10. The VH may comprise an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence SEQ ID NO: 10. VH may comprise the amino acid sequence SEQ ID NO: 10.

The antibody or antigen-binding fragment thereof may comprise a VH comprising an amino acid sequence that is at least 90% identical to the amino acid sequence SEQ ID NO: 12. The VH may comprise an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence SEQ ID NO: 12. VH may comprise the amino acid sequence SEQ ID NO: 12.

The antibody or antigen-binding fragment may comprise a VL comprising an amino acid sequence that is at least 90% identical to the amino acid sequence SEQ ID NO: 1. The VL may comprise an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence SEQ ID NO: 1. VL may comprise the amino acid sequence SEQ ID NO: 1.

The antibody or antigen-binding fragment may comprise a VL comprising an amino acid sequence that is at least 90% identical to the amino acid sequence SEQ ID NO: 5. The VL may comprise an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence SEQ ID NO: 5. VL may comprise the amino acid sequence SEQ ID NO: 5.

The antibody or antigen-binding fragment thereof may comprise a VH comprising an amino acid sequence at least 90% identical to the amino acid sequence SEQ ID NO: 17. The VH may comprise an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence SEQ ID NO: 17. VH may comprise the amino acid sequence SEQ ID NO: 17.

The antibody or antigen-binding fragment thereof may comprise a VH comprising an amino acid sequence at least 90% identical to the amino acid sequence SEQ ID NO: 18. The VH may comprise an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence SEQ ID NO: 18. VH may comprise the amino acid sequence SEQ ID NO: 18.

The antibody or antigen-binding fragment may comprise a VL comprising an amino acid sequence that is at least 90% identical to the amino acid sequence SEQ ID NO: 13. The VL may comprise an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence SEQ ID NO: 13. VL may comprise the amino acid sequence SEQ ID NO: 13.

The antibody or antigen-binding fragment may comprise a VL comprising an amino acid sequence that is at least 90% identical to the amino acid sequence SEQ ID NO: 58. The VL may comprise an amino acid sequence that is at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the amino acid sequence SEQ ID NO: 58. VL may comprise the amino acid sequence SEQ ID NO: 58.

In one aspect, the defucosylated antibody or antigen-binding fragment thereof may comprise a heavy chain comprising: VH selected from amino acid sequences comprising SEQ ID NO: 10, SEQ ID NO: 12, VH of SEQ ID NO: 36, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56 or SEQ ID NO: 57 and further comprising an IgG1 constant domain (SEQ ID NO: 31). In one aspect, the antibody variant comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 conservative or non-conservative substitutions of the full-length heavy chain , And / or the addition and / or deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 full-length heavy chains. In another aspect, the variant has at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the full-length heavy chain % Sequence identity, and where the antibody or antigen-binding fragment specifically binds CXCR5.

The defucosylated antibody of the present invention may comprise a light chain comprising a VL, the VL comprising an amino acid sequence SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 35, SEQ ID NO: 47. SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50 or SEQ ID NO: 51, wherein the antibody further comprises a light chain constant domain. As described more fully elsewhere herein, the defucosylated antibody light chain constant domain may be selected from Cκ or Cλ constant regions, such as the Cκ constant region of SEQ ID NO: 30. In one aspect, the antibody variant comprises conservative or non-conservative substitutions of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 full-length light chains , And / or the addition and / or deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 full-length light chains. In another aspect, the variant has at least 65%, at least 75%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the full length light chain % Sequence identity, and where the antibody or antigen-binding fragment specifically binds CXCR5.

The antibody or antigen-binding fragment may comprise HC, which comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or SEQ ID NO: 29 of the amino acid sequence 99% identical amino acid sequence. The HC may comprise the amino acid sequence SEQ ID NO: 29. Preferably, the antibody is defucosylated.

The antibody or antigen-binding fragment may comprise an LC comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO: 28 Amino acid sequence. The LC may comprise the amino acid sequence SEQ ID NO: 28. Preferably, the antibody is defucosylated.

Preferably, the antibody or antigen-binding fragment of the present invention is defucosylated. Even more preferably, the antibody or antigen-binding fragment thereof is defucosylated and exhibits an improved ADCC effector function as compared to an antibody that is otherwise the same as fucosylated.
Germline replacement

In certain embodiments, the antibody or antigen-binding fragment thereof comprises the following heavy chain CDR sequence: (i) CDR-H1 comprising SEQ ID NO: 7, CDR-H2 comprising SEQ ID NO: 8, and SEQ ID NO : 9 or CDR-H3 of SEQ ID NO: 11; and / or (ii) the following light chain CDR sequence: CDR-L1 comprising SEQ ID NO: 2; CDR-L2 comprising SEQ ID NO: 3; and comprising SEQ ID NO: 4 of CDR-L3. These are mouse CDRs and are preferably grafted or otherwise added in the case of human VH and VL domains. Various receptor human germline sequences are available and used to "humanize" non-human species antibody production processes in the human body will be well known in the art and discussed elsewhere herein. Therefore, skilled artisans will understand that the above mouse CDR sequences can be placed in the case of human V domain amino acid sequences. In this way, the sequence of the human germline of the recipient is generally changed to preserve antibody binding and other desired characteristics of the original parent (ie, donor) antibody. Both the CDR and the framework area (FW) can be engineered as follows.

In certain embodiments, relative to the amino acid sequence SEQ ID NO: 2, no more than 11, or no more than 10, no more than 9, no more than 8, no more than 7 are performed in the CDR-L1 , No more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 replacement. In certain embodiments, relative to the amino acid sequence SEQ ID NO: 3, no more than 6, no more than 5, no more than 4, no more than 3, no more than 3, in CDR-L2, No more than 2 or no more than one replacement. In certain embodiments, relative to the amino acid sequence SEQ ID NO: 4, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, in the CDR-L3, No more than 3, no more than 3, no more than 2 or no more than one replacement. In certain embodiments, relative to the amino acid sequence SEQ ID NO: 7, no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, in the CDR-H1, No more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 replacement. In some embodiments, with respect to the amino acid sequence SEQ ID NO: 8, no more than 17, no more than 16, no more than 15, no more than 14, no more than 13, no more than More than 12, no more than 11, no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no Replace more than 2, or not more than 1. In some embodiments, no more than 12, no more than 11, or no more than 10 are performed in CDR-H3 relative to the amino acid sequence SEQ ID NO: 9 or relative to the amino acid sequence SEQ ID NO: 11 , No more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 replacement. In some embodiments, the substitution does not change the binding affinity (K _D ) value by more than 1000 times, more than 100 times, or 10 times. In certain embodiments, the substitution is a conservative substitution according to Table 1.

In some embodiments, the substitution is a human germline substitution, in which the (donor) CDR residues are replaced by corresponding human germline (acceptor) residues to increase the human amino acid content and possibly reduce the immunogen of the antibody As described in, for example, US Patent Application Publication No. 2017/0073395 and Townsend et al., 2015, Proc. Nat. Acad. Sci. USA 112 (50): 15354-15359). For example, if the human germline DPK9 framework is used and the exemplary antibody mouse or humanized (XC154) 11G2 VL is compared, then the 11G2 VL (mouse and humanized XC154) antibody (SEQ ID NO: 2) and human germline The alignment of CDR-L1 of DPK9 is as follows:

For amino acid position number 28 (italic), the human germline residue (acceptor) and the corresponding 11G2 VL (XC154) residue (donor) are the same, and germline substitution may occur. For positions 27, 29, 30, 31, 32, 33, 34, and 35 (bold and underlined), the human germline (acceptor) residue and the corresponding 11G2 VL (XC154) (donor) residue are different. The residues of 11G2 VL (XC154) at these positions can be replaced by the corresponding human germline DPK9 residues to further increase the content of human residues. Each heavy and light chain CDR can follow the same process to increase the content of human amino acid residues while minimizing the content of mouse residues while preserving binding characteristics (such as epitope binding, affinity and the like) , Thereby reducing any possible immunogenicity against antibodies in humans, such as the human anti-mouse antibody (HAMA) immune response.

Methods and libraries for introducing human germline residues into antibody CDRs are described in detail in US Patent Application Publication No. 2017/0073395 and Townsend et al., 2015, Proc. Natl. Acad. Sci. USA. 112 (50) : 15354-15359, and both are incorporated herein by reference in their entirety.

The antibody or antigen-binding fragment thereof may comprise a VH framework, which comprises human germline VH framework sequences. The VH framework sequence may be from the human VH3 germline, VH1 germline, VH5 germline or VH4 germline. The preferred human germline heavy chain framework is derived from the framework of the VH1, VH3 or VH5 germline. For example, the VH framework from the following germline can be used: IGHV3-23, IGHV3-7, or IGHV1-69 (the germline name system is based on the IMGT germline definition). The preferred human germline light chain framework is derived from the framework of the VK or V1 germline. For example, the VL framework from the following germline can be used: IGKV1-39 or IGKV3-20 (the germline name system is based on the IMGT germline definition). Alternatively or additionally, the framework sequence may be a human germline common framework sequence, such as the framework of the following: human Vl1 common sequence, VK1 common sequence, VK2 common sequence, VK3 common sequence, VH3 germline common sequence, VH1 germline Common sequence, VH5 germline common sequence, or VH4 germline common sequence. Sequences of the human germline framework can be obtained from various public databases, such as V-base, IMGT, NCBI, or Abysis.

The antibody or antigen-binding fragment thereof may comprise a VL framework, which comprises human germline VL framework sequences. The VL framework may contain one or more amino acid substitutions, additions, or deletions, while still retaining functional and structural similarities to the germline from which the VL framework is derived. In some aspects, the VL framework and the human germline VL framework sequence are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% , At least 99% or 100% consistent. In some aspects, the antibody or antigen-binding fragment thereof comprises a VL framework comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amine groups relative to the human germline VL framework sequence Acid substitution, addition or deletion. In some aspects, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions or deletions are only in the framework region. In other aspects, the% identity is based on similarity to VL that does not include other fragments defined herein as CDRs.

The human reproductive system VL framework may be the framework of DPK9 (IMGT name: IGKV1-39). The human reproductive system VL framework may be the framework of DPK12 (IMGT name: IGKV2D-29). The human reproductive system VL framework may be the framework of DPK18 (IMGT name: IGKV2-30). The human reproductive system VL framework may be the framework of DPK24 (IMGT name: IGKV4-1). The human reproductive system VL framework may be the framework of HK102_V1 (IMGT name: IGKV1-5). The human reproductive system VL framework may be the framework of DPK1 (IMGT name: IGKV1-33). The human reproductive system VL framework may be the framework of DPK8 (IMGT name: IGKV1-9). The human reproductive system VL framework may be the framework of DPK3 (IMGT name: IGKV1-6). The human reproductive system VL framework may be the framework of DPK21 (IMGT name: IGKV3-15). The human reproductive system VL framework may be the framework of Vg_38K (IMGT name: IGKV3-11). The human reproductive system VL framework may be the framework of DPK22 (IMGT name: IGKV3-20). The human reproductive system VL framework may be the framework of DPK15 (IMGT name: IGKV2-28). The human reproductive system VL framework may be the framework of DPL16 (IMGT name: IGLV3-19). The human reproductive system VL framework may be the framework of DPL8 (IMGT name: IGLV1-40). The human reproductive system VL framework may be the framework of V1-22 (IMGT name: IGLV6-57). The human reproductive system VL framework may be the framework of the common sequence of human V1. The human reproductive system VL framework may be a framework of human Vl1 common sequence. The human reproductive system VL framework may be a framework of human V13 common sequence. The human reproductive system VL framework may be a framework of human VK common sequence. The human reproductive system VL framework may be the framework of human VK1 common sequence. The human reproductive system VL framework may be a framework of human VK2 common sequence. The human reproductive system VL framework may be a framework of human VK3 common sequence.

In some aspects, the VL framework is DPK9. Other similar framework regions for the delivery of advantageous antibodies of the invention are also predicted, which include CDRs of SEQ ID NOs: 2, 3, 4, 7, 8, 9, 11, 14, 15, 16, and the following VL amino acid sequence Specified CDR: 1, 5, 13, 28, 35, 37, 39, 47, 48, 48, 50, 51, 58, 59, 60, 61, 62, 97, 98, including DPK5, DPK4, DPK1, IGKV1 -5 * 01, DPK24, DPK21, DPK15, IGKV1-13 * 02, IGKV1-17 * 01, DPK8, IGKV3-11 * 01, and DPK22, which include FW areas 99, 97, 97, and DPK-9, respectively, 96, 80, 76, 66, 97, 97, 96, 76 and 74% identity and amino acid differences (Kabat numbering) in one or less common structural features (A) located directly in the CDR (cursor area) Lower residues, L2, L4, L35, L36, L46, L47, L48, L49, L64, L66, L68, L69, L71, (B) VH / VL chain filling residues: L36, L38, L44, L46 L87 and (C) Typical CDR structure carrier residues L2, L48, L64, L71 (see Lo, `` Antibody Humanization by CDR Grafting '', (2004) Antibody Engineering, Volume 248, Methods in Molecular Biology pages 135-159 And O'Brien and Jones, "Humanization of Monoclonal Antibodies by CDR Grafting", (2003) Recombinant Antibodies for Cancer Therapy, Vol. 207, Methods in Molecular Biology pages 81-100). Especially preferred are the framework regions of DPK5, DPK4, DPK1, IGKV1-5 * 01, DPK24, DPK21, and DPK15 that share 99, 97, 97, 96, 80, 76, 66% consistency with DPK9, respectively. Amino acid differences in these common structural features. In other aspects, the% identity is based on similarity to VL that does not include other fragments defined herein as CDRs.

The antibody or antigen-binding fragment thereof may comprise a VH framework, which comprises human germline VH framework sequences. The VH framework can include one or more amino acid substitutions, additions, or deletions while still retaining functional and structural similarities to the reproductive system from which the VL framework is derived. In some aspects, the VH framework and the human germline VH framework sequence are at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% , At least 99% or 100% consistent. In some aspects, the antibody or antigen-binding fragment thereof comprises a VH framework, which comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amine groups relative to the human germline VH framework sequence Acid substitution, addition or deletion. In some aspects, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions, additions or deletions are only in the framework region. In some aspects,% identity is based on similarity to VHs that do not include other fragments defined herein as CDRs.

The human reproductive system VH framework may be the framework of DP54 or IGHV3-7. The human reproductive system VH framework may be the framework of DP47 or IGHV3-23. The human reproductive system VH framework may be the framework of DP71 or IGHV4-59. The human reproductive system VH framework may be the framework of DP75 or IGHV1-2_02. The human reproductive system VH framework may be the framework of DP10 or IGHV1-69. The human reproductive system VH framework may be the framework of DP7 or IGHV1-46. The human reproductive system VH framework may be the framework of DP49 or IGHV3-30. The human reproductive system VH framework may be the framework of DP51 or IGHV3-48. The human reproductive system VH framework may be the framework of DP38 or IGHV3-15. The human reproductive system VH framework may be the framework of DP79 or IGHV4-39. The human reproductive system VH framework may be the framework of DP78 or IGHV4-30-4. The human reproductive system VH framework may be the framework of DP73 or IGHV5-51. The human reproductive system VH framework may be the framework of DP50 or IGHV3-33. The human reproductive system VH framework may be the framework of DP46 or IGHV3-30-3. The human reproductive system VH framework may be the framework of DP31 or IGHV3-9. The human reproductive system VH framework may be a framework of common sequences of human VH reproductive systems. The human reproductive system VH framework may be a framework of common sequences of human VH3 reproductive systems. The human reproductive system VH framework may be a framework of common sequences of human VH5 reproductive systems. The human reproductive system VH framework may be a framework of common sequences of human VH1 reproductive systems. The human reproductive system VH framework may be a framework of common sequences of human VH4 reproductive systems.

In some aspects, the VH framework is DP-54. Other similar framework regions for the delivery of advantageous antibodies of the invention are also predicted, which include CDRs of SEQ ID NOs: 7, 8, 9, 11, 19, 20, 21; and CDRs designated by the following VH amino acid sequences: 6, 10, 12, 17, 18, 36, 38, 40, 52, 53, 54, 55, 56, 57, 63, 96, including DP-50, IGHV3-30 * 09, IGHV3-30 * 15, IGHV3-48 * 01, DP-77, DP-51, IGHV3-66 * 01, DP-53, DP-48, IGHV3-53 * 01, IGHV3-30 * 02, and DP-49, which include FW area 93, 92, 92, 99, 97, 97, 96, 96, 94, 94, 93, 92% consistency and amino acid differences in one or less common structural features (Kabat numbering) (A ) Residues directly under the CDR (cursor region), H2, H47, H48 and H49, H67, H69, H71, H73, H93, H94, (B) VH / VL chain filling residues: H37, H39, H45, H47, H91, H93 and (C) typical CDR structure carrier residues H24, H71, H94 (see Lo 2004, and O'Brien and Jones 2003). Particularly preferred are the framework regions of DP-50, IGHV3-30 * 09, IGHV3-30 * 15, which share 93, 92 and 92% consistency with DP-54, respectively, and there is no amine in these common structural features Base acid difference. In some aspects,% identity is based on similarity to VHs that do not include other fragments defined herein as CDRs.

In certain embodiments, the antibody or antigen-binding fragment described herein comprises (i) VH, the VH comprising at least 50%, at least 60%, at least 70%, at least 50%, amino acid sequence SEQ ID NO: 8 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or 100% identical amino acid sequence, and / or (ii) VL, which contains at least 50%, at least 60%, at least 66%, at least 70%, at least 75% of the amino acid sequence SEQ ID NO: 1 , At least 76%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical amino acid sequence. The invention also covers any combination of these VL and VH sequences.

In certain embodiments, the antibodies or antigen-binding fragments described herein comprise (i) HC, which comprises at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% Or 100% identical amino acid sequence, and / or (ii) LC, the LC comprising at least 50%, at least 60%, at least 70%, at least 75%, at least 80% of the amino acid sequence SEQ ID NO: 28 , At least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% consistent amines Acid sequence. The invention also covers any combination of these HC and LC sequences.

In certain embodiments, the antibodies or antigen-binding fragments described herein comprise an Fc domain. The Fc domain may be derived from IgA (eg IgA ₁ or IgA ₂ ), IgG, IgE or IgG (eg IgG ₁ , IgG ₂ , IgG ₃ or IgG ₄ ).

The invention also provides antibodies or antigen-binding fragments thereof that compete with any of the antibodies or antigen-binding fragments described herein, such as any of the antibodies (or antigen-binding fragments) provided herein For human CXCR5. For example, if the binding of an antibody or antigen-binding fragment thereof to human CXCR5 prevents subsequent binding of human CXCR5 to CXCL13, the antibody or antigen-binding fragment competes with CXCL13 for human CXCR5 binding.

The antibodies and antigen-binding fragments provided by the present invention include monoclonal antibodies, multiple antibodies, antibody fragments (such as Fab, Fab ', F (ab') ₂ , Fv, Fc, etc.), chimeric antibodies, bispecific antibodies, Binding antibodies, single chain (scFv), mutants thereof, fusion proteins containing antibody parts, domain antibodies (dAbs), humanized antibodies, and any other immunoglobulin molecules containing antigen recognition sites of desired specificity Modified configurations include glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. Antibodies and antigen-binding fragments can be mouse, rat, human, or any other source (including chimeric or humanized antibodies). In some embodiments, anti-systemic monoclonal antibodies. In some embodiments, the anti-systemic chimeric, humanized, or human antibodies. In certain embodiments, the anti-systemic human antibody. In certain embodiments, the anti-systemic humanized antibody.
Epitope localization

The invention also provides an antibody or antigen-binding fragment thereof that binds to the same human CXCR5 epitope as the antibody or antigen-binding fragment described herein. For example, antibody competition analysis (and overlapping epitope analysis) can be assessed by SPR, flow cytometry, and any other analysis known in the art.

In one aspect, the invention encompasses antibodies or antigen-binding fragments thereof that bind human CXCR5 and cynomolgus monkey CXCR5, but not mouse CXCR5.

The present invention also includes antibodies or antigen-binding fragments that bind to the N-terminal region ("N") of human CXCR5. Based on the numbering of the amino acid sequence SEQ ID NO: 31, the N-terminus of CXCR5 contains amino acid residue numbers 1-51.

The present invention includes an antibody or antigen-binding fragment thereof, which specifically binds to an epitope containing leucine (L) at amino acid residue position number 11 according to the amino acid sequence SEQ ID NO: 32 CXCR5.

In one aspect, the invention includes an antibody or antigen-binding fragment thereof according to the amino acid sequence of SEQ ID NO: 32, which contains aspartic acid (D) at position 22 of the amino acid residue The epitope specifically binds CXCR5.

In one aspect, the invention includes an antibody or antigen-binding fragment thereof according to the amino acid sequence SEQ ID NO: 32, which contains the amino acid residue and amino acid residue at position 11 of the amino acid residue The antigenic determination of aspartic acid at base position number 22 specifically binds to CXCR5.

The present invention includes antibodies or antigen-binding fragments that specifically bind to CXCR5, wherein the amino acid residue at position 11 is leucine according to the amino acid sequence SEQ ID NO: 32.

The present invention includes antibodies or antigen-binding fragments according to the amino acid sequence SEQ ID NO: 32, which specifically binds CXCR5 when the amino acid residue at position 11 is leucine, but at the position When the amino acid residue at number 11 is threonine, CXCR5 is not bound.

In one aspect, the invention includes an antibody or antigen-binding fragment thereof that specifically binds to wild-type human CXCR5 but is numbered according to the amino acid sequence SEQ ID NO: 32, amino acid residue position number 11 is not white amine Does not bind in the case of acid.

The present invention includes antibodies or antigen-binding fragments that specifically bind to CXCR5, wherein the amino acid residue at position 22 is aspartic acid according to the amino acid sequence SEQ ID NO: 32.

In one aspect, the invention includes an antibody or antigen-binding fragment thereof that specifically binds to wild-type human CXCR5 but the amino acid residue at position 22 according to the amino acid sequence SEQ ID NO: 32 is not In the case of aspartic acid, it does not bind.

In one aspect, the invention includes antibodies or antigen-binding fragments thereof that specifically bind to wild-type human CXCR5 but are numbered 22 based on the amino acid sequence SEQ ID NO: 32, amino acid residue position number 22 alanine The case is not combined.

The present invention includes antibodies or antigen-binding fragments that specifically bind to CXCR5, wherein the amino acid residue at position 11 is leucine and the amine at position 22 according to the amino acid sequence SEQ ID NO: 32 The acid residue is aspartic acid.

In one aspect, the invention includes an antibody or antigen-binding fragment thereof that specifically binds to human CXCR5 but does not bind to CXCR5 under the following conditions: according to the amino acid sequence SEQ ID NO: 32, the amino group at position 11 The acid residue is not leucine and the amino acid residue at position 22 is not aspartic acid.

In one aspect, the invention includes an antibody or antigen-binding fragment thereof that specifically binds to human CXCR5 but does not bind under the following conditions: according to the amino acid sequence number of SEQ ID NO: 32, the amino group at position number 11 The acid residue is threonine and the amino acid residue at position 22 is alanine.

Those skilled in the art will understand that according to the amino acid sequence SEQ ID NO: 32, amino acid residues at position number 11 and / or position number 22 are crucial for the binding of the antibody of the present invention according to the amino acid sequence SEQ ID NO: 32 important. More specifically, these amino acid residues are essential for the binding of CXCR5 to antibody 11G2 or its antigen-binding fragments. Therefore, skilled artisans will understand that the present invention includes antibodies or antigen-binding fragments that specifically bind to CXCR5, wherein the amino acid residue at position 11 is leucine and the amino acid residue at position 22 is aspart Amino acid. The substitution or deletion of these amino acid residues will result in loss of binding to CXCR5. Some substitutions of the amino acid residue at position 11 can preserve the binding, but the substitution of amino acid residues of leucine (leucine) and threonine is not. Similarly, some substitutions or deletions of the amino acid residue at position 22 can preserve the binding, but the substitution of aspartic acid and alanine at the amino acid residue number is not. Therefore, the 11G2 antibody or antigen-binding fragment thereof, or an antibody that competes with it is characterized in that the antibody binds to human CXCR5, in which leucine 11 and aspartic acid 22 are present according to the amino acid sequence SEQ ID NO: 32. However, the antibody does not bind when leucine 11 is replaced with threonine or another amino acid residue, and the antibody does not bind when aspartate 22 is replaced with alanine or another amino acid residue Combine. After amino acid substitution at amino acid residue position number 11 and / or number 22, the test for binding loss can be performed using various methods well known in the art, including binding analysis using point mutation polypeptides, as described herein Illustrated method.

Based on the teaching content provided herein, those skilled in the art will understand that the antibody of the present invention can compete with, for example, 11G2, but does not include the following epitope: it is based on the amino acid sequence SEQ ID NO: 32 number, including Leucine at amino acid residue number 11 and / or aspartic acid at amino acid residue number 22. That is, the antibody can compete with the antibody of the present invention for binding to CXCR5, but the competing antibody binds to alanine acid at amino acid residue number 11 or aspartic acid at amino acid residue number 22 via a different amine Basic acids (such as the substitution of threonine for leucine and / or the substitution of alanine for aspartic acid), such as 2C9, 11A7 and 16D7, are not affected. Therefore, the antibody of the present invention competes for binding to CXCR5 and when amino acid residue number 11 is not leucine, and more specifically it is threonine, and / or when amino acid residue number 22 is not Aspartic acid, and more specifically in the case of alanine, does not bind CXCR5. As previously stated elsewhere herein, the production of mutant CXCR5 proteins and the analysis used to assess the competitive binding of antibodies are well known in the art, including their methods described herein. Therefore, based on the teaching content provided herein, skilled artisans will be able to easily recognize the following antibodies: they bind CXCR5, compete with the antibodies of the present invention for binding, and in certain amino acids such as leucine 11, aspartic acid 22 or the loss of the ability to bind CXCR5 mutant protein in the case of substitution, all according to the amino acid sequence SEQ ID NO: 32 numbering.

In another aspect, the invention includes an antibody or antigen-binding fragment thereof that competes for binding with antibody 11G2 or an antigen-binding fragment thereof, wherein the antibody is numbered according to the amino acid sequence SEQ ID NO: 32, the amino acid residue No. 11 does not bind CXCR5 when it is not leucine.

In another aspect, the invention includes an antibody or antigen-binding fragment thereof that competes for binding with antibody 11G2 or an antigen-binding fragment thereof, wherein the antibody is numbered according to the amino acid sequence SEQ ID NO: 32, the amino acid residue No. 22 does not bind CXCR5 when it is not leucine.

In another aspect, the invention includes an antibody or antigen-binding fragment thereof that competes for binding with antibody 11G2 or an antigen-binding fragment thereof, wherein the antibody is numbered according to the amino acid sequence SEQ ID NO: 32, the amino acid residue When the number 11 is not leucine and the amino acid residue number 22 is not aspartic acid, CXCR5 is not bound.

In another aspect, the invention includes an antibody or antigen-binding fragment thereof that competes for binding with antibody 11G2 or an antigen-binding fragment thereof, wherein the antibody is numbered according to the amino acid sequence SEQ ID NO: 32, the amino acid residue No. 11 is not bound to CXCR5 in the case of threonine.

In another aspect, the invention includes an antibody or antigen-binding fragment thereof that competes for binding with antibody 11G2 or an antigen-binding fragment thereof, wherein the antibody is numbered according to the amino acid sequence SEQ ID NO: 32, the amino acid residue No. 22 is not bound to CXCR5 in the case of alanine.

In another aspect, the invention includes an antibody or antigen-binding fragment thereof that competes for binding with antibody 11G2 or an antigen-binding fragment thereof, wherein the antibody is numbered according to the amino acid sequence SEQ ID NO: 32, the amino acid residue Number 11 is threonine, and amino acid residue number 22 is alanine, and CXCR5 is not bound.
Biological activity of anti- CXCR5 antibody

In addition to binding to epitopes on CXCR5, the antibodies or antigen-binding fragments of the invention can also mediate biological activity. That is, the invention includes an isolated antibody or antigen-binding fragment thereof that specifically binds CXCR5 and mediates at least one detectable activity selected from: (a) binds CXCR5 + cells with high apparent affinity, but does not bind to express CXCR5 Cells of mouse, rat or rabbit heterologs; (b) antagonize the inhibition of CXCL13 release of cAMP triggered by forskolin; (c) trigger CXCR5 in human donors and cynomolgus monkey PBMC and human donor TMC ADCC representing cells; (d) binding to human CXCR5 but not to human chemokine receptors CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CMKLR1, CXCR3R1, CXCR1, CXCR2, CXCR3 , CXCR4, CXCR6, CXCR7, or XCR1; (e) B cell depletion in peripheral blood and / or peripheral lymphoid organs (ie, lymph nodes, spleen, Peyer ’s patches, and mucosa-associated lymphoid tissue); (f) depletion Tfh-like cells in the blood; (g) depleting bona fide Tfh cells in surrounding lymphoid organs; and (h) impairing the humoral immune memory response.

In one aspect, the present invention includes antibodies, or binds CXCR5 + cells with high apparent affinity, but does not bind cells expressing CXCR5 mouse, rat, or rabbit xenologs. Flow cytometry can be used to assess apparent affinity binding to detect the binding of antibodies to cells expressing the target protein (eg CXCR5). Cells can be transiently or stably transfected with a nucleic acid encoding CXCR5. Alternatively, the cell may be a cell that naturally expresses CXCR5 on its surface. Regardless of the source of the CXCR5 + cell, a variety of methods recognized in this technology can be used to easily assess the binding of the antibody to the cell. The antibody or antigen-binding fragment thereof binds human CXCR5 or cynomolgus cynomolgus monkey CXCR5, but does not detectably bind to mouse, rat or rabbit CXCR5, or to a much lesser extent.

The invention includes antibodies or antigen-binding fragments thereof that specifically bind to CXCR5 and antagonize the activity mediated by the binding of CXCL13 to CXCR5. There are many known in the art for determining the inhibition of CXCR5-CXCL13 signaling mediated activity. One such analysis is the cAMP report analysis. In such analysis, forskolin induces cAMP production, which is inhibited by CXCR5-CXCL13 signaling in cells that stably express CXCR5. Therefore, in the presence or absence of antibodies, the ability of anti-CXCR5 antibodies to bind to CXCR5 and antagonize CXCR5-CXCL13 signaling was assessed by measuring the level of cAMP produced. Preferably, the antibody may be about 50 pM, about 100 pM, about 200 pM, about 400 pM, about 600 pM, about 700 pM, about 750 pM, about 790 pM, about 800 pM, about 850 pM, about 900 pM, An EC50 of about 950 pM, about 960 pM, about 970 pM, about 980 pM, about 990 pm, or about 1000 pM mediates a dose-dependent increase in cAMP levels. More preferably, the antibody or antigen-binding fragment thereof inhibits forskolin-triggered cAMP with an EC50 of about 961 pM, resulting in CXCL13 inhibition.

The present invention includes antibodies or antigen-binding fragments thereof that specifically bind to CXCR5 and trigger ADCC of CXCR5 expressing cells in human donor and cynomolgus monkey PBMC and human donor tonsil monocytes (TMC). Many ADCC assays can be used to assess the ADCC activity of antibodies. One such exemplary analysis is described herein in Examples 6 and 7, but the invention is not limited to this particular ADCC analysis. The present invention includes antibodies or antigen-binding fragments thereof that exhibit an EC50 of about 0.11 pM, 0.2 pM, 0.5 pM, 1 pM. 1.5 pM, 2.0 pM, 2.5 pM, 3.0 pM, 4.5 pM, 4.8 pM, 5.0 pM, 6.0 pM , 7.0 pM, 8.0 pM, 9.0 pM, 10 pM, 11 pM, 12 pM, 15 pM, 20, pM, 25 pM, 30 pM, 35 pM, or 40 pM for human B cells, human Tfh-like cells, human ADCC activity of Tfh cells and cynomolgus macaque B cells. More preferably, the antibody or antigen-binding fragment exhibits an EC50 of about 2.01 ± 2.28 pM for human B cells, about 4.82 ± 2.88 pM for human Tfh-like cells, about 0.11 pM for human THF cells, and about 15.3 ± 11.7 pM for ADCC activity of cynomolgus macaque B cells. Even more preferably, the antibody or antigen-binding fragment thereof is defucosylated.

The present invention covers the following antibodies or antigen-binding fragments thereof that bind human CXCR5 but do not detectably bind human proteins CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CMKLR1, CXCR3R1, CXCR1 , CXCR2, CXCR3, CXCR4, CXCR6, CXCR7 or XCR1.

The present invention covers antibodies or antigen-binding fragments thereof that specifically bind to CXCR5 and exhibit dose-dependent depletion of B cells in peripheral blood. The depletion may be permanent, or more preferably, the depletion is temporary and / or reversible. In one aspect, the dose of antibody sufficient to mediate B cell depletion may range from 0.001 to about 0.2 mg, which is 0.0001 mg compared to the percentage of B cells or Tfh-like cells in the peripheral blood that did not receive the antibody / kg can mediate about 50% depletion of B cells and Tfh-like cells in the peripheral blood of cynomolgus monkeys. In addition, antibodies can mediate the maximum depletion of B cells and Tfh-like cells in the peripheral blood of cynomolgus cynomolgus monkeys administered at a dose of less than 5 mg / kg (intravenous administration (IV)). Preferably, part of the B cells, Tfh-like and Tfh cells appear to completely recover after the antibody is administered.

The present invention includes antibodies or antigen-binding fragments thereof that specifically bind to CXCR5 and impair humoral immune memory responses. In one aspect, humoral memory responses are assessed using vaccine recall analysis. That is, antibodies are administered to individuals who have previously been vaccinated with an antigen (eg, tetanus toxoid (TT)). The individual is administered the antigen a second time and the immune response (eg, IgM and IgG titers) is compared to the immune response of the individual who has not otherwise been administered the antibody otherwise. After providing the teachings disclosed herein, the skilled artisan will understand that many assays for measuring humoral memory responses can be used to assess the ability of antibodies or antigen-binding fragments to impair humoral memory responses. In addition, the skilled artisan will understand that the ability to impair humoral memory response is a desired characteristic of an antibody or antigen-binding fragment thereof to be used as a therapeutic agent to treat or prevent an immune disease in an individual in need.

The present invention encompasses antibodies or antigen-binding fragments thereof that exhibit at least one, preferably two, even better three, even better four, yet better five, even better six, and even better seven discussed above Biological activity, and even better all the biological activities discussed above.
III . Defucosylated anti- CXCR5 antibody

In one aspect, antibodies having a carbohydrate structure that lacks fucose attached (directly or indirectly) to the Fc region (ie, defucosylated antibodies) are provided. For example, the amount of fucose in a composition comprising a plurality of these antibodies may be from 0 percent to about 30 percent, 0 percent to about 20 percent, 0 percent to about 15 percent, 0 percent to about 10 percent, and More preferably from 0 percent to about 5 percent. In some embodiments, a composition comprising a plurality of such antibodies comprises at least 80%, more preferably at least 85%, still more preferably at least 90%, even more preferably at least 95% defucosylated antibody, still more preferably At least 99%, and most preferably at least 99.5% defucosylated antibodies. In some embodiments, the antibody is 100% defucosylated; that is, no fucose was detected at Asn297 using methods recognized in the art for detecting fucose in antibodies. The amount of fucose is determined by calculating the average amount of fucose in the sugar chain at Asn297 relative to the sum of all sugar structures attached to Asn 297 (eg, compound, hybrid, and high mannose structures).

In some embodiments, the content of fucosylation does not exceed 0.5%, which is based on the limit of detection (LOQ) of the test method. Therefore, in some embodiments, the content of fucosylation is greater than or equal to 99.5%. The N-linked oligosaccharide profile method can be used to determine the content of fucosylation, sialylation, mannosylation and terminal galactosylation in the sample. N-linked glycans can be evaluated using the N-linked oligosaccharide method. Briefly, N-linked glycans are enzymatically released from the protein by peptide-N-glycosidase F. The glycans were then derived by fluorescent agents and analyzed using hydrophilic interaction liquid chromatography and fluorescent detection. The chromatogram of the reference material is then compared with the reference material. This method and many other methods are known in the art for assessing the fucosylation of antibodies, and it can be used to determine the level of fucosylation present in the antibodies of the present invention.

Non-limiting exemplary methods for detecting fucose in antibodies include MALDI-TOF mass spectrometry (see, for example, WO 2008/077546), and HPLC measurement of the fluorescently labeled oligosaccharides released (see, for example, Schneider et al., "N-Glycan analysis of monoclonal antibodies and other glycoproteins using UHPLC with fluorescence detection", Agilent Technologies, Inc. (2012); Lines, J. Pharm. Biomed. Analysis, 14: 601-608 (1996); Takahasi, J. Chrom., 720: 217-225 (1996)), capillary electrophoresis measurement of the fluorescently labeled oligosaccharides released (see for example Ma et al., Anal. Chem., 71: 5185-5192 (1999)) , And HPLC using pulsed current detection to measure monosaccharide compositions (see, for example, Hardy et al., Analytical Biochem., 170: 54-62 (1988)). Asn297 refers to asparagine residues (EU numbering of Fc region residues) located at about position 297 in the Fc region; however, Asn297 can also be located about ± 3 amino acids upstream or downstream of position 297, and That is, between positions 294 and 300, it is due to a slight sequence deviation of the antibody. In the CXCR5 antibody described herein, Asn297 was found in the sequence QYNST, and it is bold and underlined in Table 16 (eg, SEQ ID NO: 31 of the wild-type human IgG1 Fc domain).

Fucosylated variants can have improved ADCC function. See, for example, US Patent Publication No. US 2003/0157108 (Presta, L.); US 2004/0093621 (Kyowa Hakko Kogyo Co., Ltd). Examples of publications regarding "defucosylated" or "fucose-deficient" antibodies include: US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al. J. Mol. Biol. 336: 1239-1249 (2004); Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004). Examples of cell lines capable of producing defucosylated antibodies include Lec 13 CHO cells lacking protein fucosylation (Ripka et al. Arch. Biochem. Biophys. 249: 533-545 (1986); US Patent Application No. US 2003/0157108 Al, Presta, L; and WO 2004/056312, Adams et al., Especially Example 11), and gene knockout cell lines, such as α-1,6-fucosyltransferase gene, FUT8, Gene knockout CHO cells (see, for example, Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94 (4): 680-688 (2006); and WO2003 / 085107).

The antibody further has a bisecting oligosaccharide, for example, a bi-touch oligosaccharide attached to the Fc region of the antibody is bisected by GlcNAc. Such antibodies may have reduced fucosylation and / or improved ADCC function. Examples of such antibodies are described in, for example, WO 2003/011878 (Jean-Mairet et al.); US Patent No. 6,602,684 (Umaña et al.); And US 2005/0123546 (Umaña et al.). An antibody in which at least one galactose residue in the oligosaccharide is linked to the Fc region is also provided. Such antibodies may have improved CDC function. Such antibodies are described in, for example, WO 1997/30087 (Patel et al.); WO 1998/58964 (Raju, S.); and WO 1999/22764 (Raju, S.).

In some embodiments of the invention, the defucosylated antibody mediates ADCC more efficiently than the parent antibody containing fucose in the presence of human effector cells. In general, ADCC activity can be determined using in vitro ADCC analysis as disclosed herein, but covers other analyses or methods for determining ADCC activity, such as animal models and the like.

In some embodiments, the defucosylated anti-CXCR5 antibody has enhanced ADCC activity in vitro and / or in vivo. In some embodiments, the defucosylated anti-CXCR5 antibody has enhanced ADCC activity in vitro. In some embodiments, in vitro ADCC activity is determined by the methods described herein. Briefly, serial dilutions of anti-CXCR5 antibodies or isotype controls were incubated with peripheral blood mononuclear cells (PBMC) from healthy human donors or cynomolgus monkeys. In this analysis, the PBMCs are the source of natural killer (NK) effector cells and target CXCR5 + B and Tfh-like cells. Flow cytometry was used to quantify the number of B cells and Tfh-like cells remaining after approximately 20 hr. The cytotoxicity titration curve was generated by plotting the percentage of cytotoxicity of the antigen-binding population to the logarithm of the PF-06835375 antibody concentration. The EC ₅₀ value was determined using GraphPad Prism □ (Version 6.0, GraphPad Software, Inc, San Diego, CA) nonlinear regression curve fitting and the sigmoid logarithm of the agonist dose-response model:
Log ( agonist ) versus response - different slopes ( four parameters )
Y = Bottom + (Top-Bottom / (1 + 10 ^ ((LogEC ₅₀ -X) * Hill slope )))
Where Y is the percentage of cytotoxicity, X is the antibody concentration, Top is the maximum Y value corresponding to the upper plateau of the S-shaped curve, Bottom is the minimum Y value corresponding to the plateau of the lower S-shaped curve (limited to 0) , And LogEC ₅₀ is the logarithm of the antibody concentration at the inflection point of the curve.

The EC ₅₀ value was summarized by experiment using the mean and standard deviation (STDEV). In some embodiments, the ability of humanized mAb to induce ADCC from bona fide Tfh cells from human tonsils is similarly evaluated using CD4 + T cells isolated from tonsil monocytes and addition of NK cells isolated from PBMC. In some embodiments, Ba / F3 cells expressing CXCR5 are used as target cells. In some embodiments, cytotoxicity is determined by quantifying LDH release using CytoTox non-radioactive cytotoxicity analysis (Promega, Madison, WI).

In some embodiments, 5% Triton X-100 is used to determine maximum dissolution and spontaneous release in the absence of antibody. In some embodiments, the percentage of specific lysis rate can be determined using the following formula: (experimental value-spontaneous release) / (maximum value-spontaneous release) × 100 = percentage of specific lysis rate. In some embodiments, at a concentration of at least one antibody tested, the specific lysis rate caused by the defucosylated anti-CXCR5 antibody with enhanced ADCC activity is greater than that caused by the same amount of fucosylated antibody It is at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 65, at least 70, or at least 75 percentage points greater than the lysis rate. In some embodiments, the specific lysis rate caused by the defucosylated anti-CXCR5 antibody with enhanced ADCC activity is at least 10, at least 15, at least 20 greater than the specific lysis rate caused by the fucosylated antibody , At least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 60, at least 65, at least 70, or at least 75 percentage points, where the concentration of each antibody is between 0.01 and 1 microg / ml and the target The cell line expresses CXCR5 Ba / F3 cells. In some embodiments, the antibody is tested at a concentration range of 0.000005 microg / ml to 5 microg / ml.

In some embodiments, the defucosylated anti-CXCR5 antibody has enhanced affinity for FcyRIIIA. In some embodiments, the defucosylated anti-CXCR5 antibody has enhanced affinity for FcyRIIIA (V158). In some embodiments, the defucosylated anti-CXCR5 antibody has enhanced affinity for FcyRIIIA (F158). In some embodiments, antibody affinity for FcγRIIIA is determined using surface plasmon resonance and / or the following method: it is described with reference to FcγRIIIA (V158), but is also suitable for determining affinity for FcγRIIIA (F158). Briefly, in some embodiments, fucosylated or defucosylated anti-CXCR5 antibodies are captured on protein A-coated polydextrose sheets. FcyRIIIA (V158) is injected at various concentrations (available from, for example, R and D systems). The association constant, dissociation constant and affinity of FcγRIIIA (V158) for fucosylated and defucosylated anti-CXCR5 antibodies can be, for example, using software with a surface plasmon resonance system (e.g. Biacore T200 evaluation software 1: 1 Combined model) determination. In some embodiments, the defucosylated anti-CXCR5 antibody may have enhanced affinity for FcγRIIIA (such as FcγRIIIA (V158) or FcγRIIIA (F158)) and may be more affinity than the fucosylated anti-CXCR5 antibody At least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 7 times, at least 10 times, at least 12 times, at least 15 times, at least 17 times, 20 times, 30 times, 50 times, 100 times, 500 A fold, or at least a 1000-fold affinity, binds to FcyRIIIA.
IV . Anti- CXCR5 antibody performance and production
Nucleic acid encoding anti- CXCR5 antibody

The invention also provides polynucleotides encoding any of the antibodies, including the antibody fragments and modified antibodies described herein. The invention also provides methods for making any of the polynucleotides described herein. Polynucleotides can be manufactured and expressed by procedures known in the art.

The sequence of the desired antibody, definition, antibody fragment or antigen-binding fragment thereof, and nucleic acid encoding the antibody or fragment thereof can be determined using standard sequencing techniques. The nucleic acid sequence encoding the desired antibody, definition, antibody fragment or antigen-binding fragment thereof can be inserted into various vectors (such as selection and expression vectors) used for recombinant production and characterization. Nucleic acids encoding heavy chains, defined antibody fragments or antigen-binding fragments of heavy chains, and nucleic acids encoding light chains, defined antibody fragments or antigen-binding fragments of light chains can be selected from the same vector or different vectors.

In one aspect, the invention provides a polynucleotide encoding the amino acid sequence of any of the following CXCR5 antibodies and antigen-binding fragments thereof: mouse 11G2 VH, mouse 11G2 VL, chimeric 11G2 VH, Chimeric 11G2 VL, h11G2 VH (XC152), h11G2 VH (XC155), h11G2 VH (XC156), h11G2 VH (XC157), h11G2 VH (XC350), h11G2 VH (XC351), h11G2 VH (XC352), h11G2 VH XC353), h11G2 VH (XC354), h11G2 VL (XC151), h11G2 VL (XC153), h11G2 VL (XC154), h11G2 VL (XC346), h11G2 VL (XC347), h11G2 VL (XC348), h11G2 VL (XC354) , Mouse 41A10 VH, mouse 41A10 VL, chimeric 41A10 VH, chimeric 41A10 VL, humanized 41A10 VH (XC147), h41A10 VH (XC148), h41A10 VH (XC150), h41A10 VL (XC142), h41A10 VL ( XC143), h41A10 VL (XC144), h41A10 VL (XC145), h41A10 VL (XC146), h41A10 VL (XC149), mouse 5H7 VH, mouse 5H7 VL, chimeric 5H7 VH, and chimeric 5H7 VL. The polynucleotide encoding the above amino acid sequence encodes at least 91%, 92%, 93%, 94%, 95%, 96% of the amino acid sequence of the antibody or antigen-binding fragment of the present invention as disclosed herein , 97%, 98% or 99% identical amino acid sequence.

The present invention provides a polynucleotide encoding an amino acid sequence of an antibody or antigen-binding fragment thereof, which binds to an epitope that is substantially the same as an antibody selected from the group consisting of: chimeric 41A10, humanized 41A10 (142/147 ), Humanized 41A10 (142/148), chimeric 11G2, fucosyl chimeric 11G2, humanized 11G2 (151/152), humanized 11G2 (153/155), humanized 11G2 (153/156), Humanized 11G2 (154/155), Humanized 11G2 (154/157) and Defucosylated Humanized 11G2 (154/155).

The present invention provides a polynucleotide encoding an amino acid sequence of an antibody or antigen-binding fragment thereof. The antibody or antigen-binding fragment competes with an antibody selected from the group consisting of the following for binding to CXCR5: chimeric 41A10, humanized 41A10 ( 142/147), humanized 41A10 (142/148), chimeric 11G2, fucosyl chimeric 11G2, humanized 11G2 (151/152), humanized 11G2 (153/155), humanized 11G2 (153/155) 156), Humanized 11G2 (154/155), Humanized 11G2 (154/157) and Defucosylated Humanized 11G2 (154/155).

The present invention provides polynucleotides encoding one or more proteins comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-29, SEQ ID NO: 35-40 and SEQ ID NO: 47-63.

The present invention provides polynucleotides comprising nucleic acid sequences as set forth in one or more of SEQ ID NO: 106, 107, 108, and 109. The present invention provides a polynucleotide comprising the nucleic acid sequence as set forth in SEQ ID NO: 95. The present invention provides a polynucleotide comprising the nucleic acid sequence as set forth in SEQ ID NO: 96. The invention provides a polynucleotide comprising the nucleic acid sequence as set forth in SEQ ID NO: 97. The present invention provides a polynucleotide comprising the nucleic acid sequence as set forth in SEQ ID NO: 98.

The present invention provides a polynucleotide comprising one or both of nucleic acid sequences of a DNA insert deposited with ATCC and having plastids with accession numbers PTA-124323 and PTA-124324.

The present invention provides a polynucleotide comprising the nucleic acid sequence of the insert deposited in the plastid with accession number PTA-124323 deposited in the ATCC.

The present invention provides a polynucleotide comprising the nucleic acid sequence of the insert deposited in the plastid with accession number PTA-124324 deposited in the ATCC.

The present invention provides a polynucleotide comprising a nucleic acid sequence of an insert deposited with ATCC and having a plastid with accession number PTA-124323 and accession number PTA-124324.

The invention provides a polynucleotide comprising the nucleic acid sequence of an insert deposited with ATCC and having a plastid with accession number PTA-124323.

The present invention provides a polynucleotide comprising a nucleic acid sequence of an insert deposited with ATCC and having a plastid with accession number PTA-124324.

The invention provides cells comprising one or more nucleic acid molecules as set forth in one or more of SEQ ID NO: 106, 107, 108, and 109. The present invention provides cells comprising one or more nucleic acid molecules as set forth in SEQ ID NO: 106 and 107. The invention provides cells comprising one or more nucleic acid molecules as set forth in SEQ ID NO: 108 and 109.

In another aspect, the present invention provides polynucleotides encoding anti-CXCR5 antibodies and variants thereof, wherein such variant polynucleotides share at least 70% with any of the specific nucleic acid sequences disclosed herein , At least 75%, at least 80%, at least 85%, at least 87%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%. At least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. These amounts are not meant to be limiting, and the increase between the percentages is specifically conceived as part of the invention.

The invention provides polypeptides encoded by the nucleic acid molecules described herein.

In one embodiment, the VH and VL domains or antigen-binding fragments thereof or full-length HC or LC are encoded by independent polynucleotides. Alternatively, both VH and VL or their antigen-binding fragments or HC and LC are encoded by a single polynucleotide.

The invention also encompasses polynucleotides complementary to any such sequences. Polynucleotides can be single-stranded (coding or antisense) or double-stranded, and can be DNA (genomic, cDNA or synthetic) or RNA molecules. RNA molecules include HnRNA molecules, which contain introns and correspond to DNA molecules in a one-to-one manner; and mRNA molecules, which do not contain introns. Additional coding or non-coding sequences may (but not necessarily) be present in the polynucleotide of the present invention, and the polynucleotide may (but not necessarily) be linked to other molecules and / or supporting materials.

Polynucleotides may include native sequences (ie, endogenous sequences encoding antibodies or portions thereof) or may include variants of such sequences. Polynucleotide variants contain one or more substitutions, additions, deletions and / or insertions so that the immunoreactivity of the encoded polypeptide is not reduced relative to natural immunoreactive molecules. The effect on the immunoreactivity of the encoded polypeptide can generally be assessed as described herein. In some embodiments, the variant exhibits at least about 70% identity with the polynucleotide sequence encoding the natural antibody or a portion thereof, in some embodiments at least about 80% identity, in some embodiments at least about 90% Consistency, and in some embodiments at least about 95% consistency. These amounts are not meant to be limiting, and the increase between the percentages is specifically conceived as part of the invention.

In the maximum correspondence alignment as described below, if the nucleotide or amino acid sequences in the two sequences are the same, the two polynucleotide or polypeptide sequences are called "identical." The comparison between two sequences is usually performed by comparing sequences in a comparison window to identify and compare the sequence similarity of local regions. As used herein, a "comparison window" refers to a fragment of at least about 20 adjacent positions, usually 30 to about 75, or 40 to about 50, wherein after the two sequences are optimally aligned, the sequences can be the same as the adjacent positions Compare the number of reference sequences.

The optimal sequence alignment for comparison can be performed using the MegAlign ^® program (DNASTAR ^® , Inc., Madison, WI) in the Lasergene ^® suite of bioinformatics software using preset parameters. This program embodies several comparison schemes described in the following references: Dayhoff, MO, 1978, A model of evolutionary change in proteins-Matrices for detecting distant relationships. In Dayhoff, MO (Editor) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Volume 5, Supplement 3, pages 345-358; Hein J., 1990, Unified Approach to Alignment and Phylogenes page 626- Page 645 Methods in Enzymology Volume 183, Academic Press, Inc., San Diego, CA; Higgins, DG and Sharp, PM, 1989, CABIOS 5: 151-153; Myers, EW and Muller W., 1988, CABIOS 4: 11-17; Robinson, ED, 1971, Comb. Theor. 11: 105; Santou, N., Nes, M., 1987, Mol. Biol. Evol. 4: 406-425; Sneath, PHA and Sokal, RR, 1973, Numerical Taxonomy the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, WJ and Lipman, DJ, 1983, Proc. Natl. Acad. Sci. USA 80: 726-730.

In some embodiments, the "percent sequence identity" is by comparing at least 20 positions of the two best aligned sequences in the comparison window, where compared to the reference sequence used to optimally align the two sequences (which No additions or deletions are included), the portion of the polynucleotide or polypeptide sequence in the comparison window may contain 20% or less, usually 5 to 15%, or 10 to 12% additions or deletions (ie gaps). The percentage is calculated by determining the number of positions where the same nucleic acid base or amino acid residue appears in both sequences to obtain the number of matching positions, and dividing the number of matching positions by the total number of positions in the reference sequence (that is, the window size ) And multiply the result by 100 to get the percent sequence identity.

The variant may also or alternatively be substantially homologous to the natural gene or a part or complementary sequence thereof. Such polynucleotide variants are capable of hybridizing to naturally-occurring DNA sequences encoding natural antibodies (or complementary sequences) under appropriately stringent conditions.

Suitable "moderately stringent conditions" include pre-washing in a solution of 5 X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridization overnight at 50 X-65 ° C and 5 X SSC; then use at 65 ° C Each of 2X, 0.5X and 0.2X SSC containing 0.1% SDS was washed twice for 20 minutes.

As used herein, "highly stringent conditions" or "higher stringency conditions" are as follows: (1) Use low ionic strength and high temperature for washing, such as 0.015 M sodium chloride / 0.0015 M sodium citrate at 50 ° C /0.1% sodium lauryl sulfate; (2) Use a denaturant during hybridization, such as formamide, for example 50% (v / v) formamide / 0.1 with 0.1% bovine serum albumin at 42 ° C % Ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate; or (3) 50% at 42 ° C Formamide, 5 × SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 × Denhardt's solution, sonicated salmon sperm DNA (50 µg / mL ), 0.1% SDS and 10% Dextran Sulfate, where at 42 ° C in 0.2 × SSC (sodium chloride / sodium citrate) and at 55 ° C in 50% formamide wash, followed by 55 Wash with a higher stringency wash consisting of 0.1 × SSC with EDTA at ℃. Skilled artisans will know how to adjust temperature, ionic strength, etc. as needed to accommodate factors such as probe length and the like.

Those of ordinary skill in the art will understand that due to the degeneracy of the genetic code, there are many nucleotide sequences encoding polypeptides as described herein. Some of these polynucleotides carry minimal homology to the nucleotide sequence of any natural gene. Nonetheless, the present invention particularly expects polynucleotides that change due to differences in codon usage. In addition, the dual genes containing the genes of the polynucleotide sequences provided herein are within the scope of the present invention. A dual gene line is an endogenous gene that changes due to one or more mutations in nucleotides, such as deletions, additions, and / or substitutions. The resulting mRNA and protein may (but need not) have varying structures or functions. Dual genes can be identified using standard techniques such as hybridization, amplification, and / or database sequence comparison.

The polynucleotide of the present invention can be obtained using chemical synthesis, recombinant methods, or PCR. Chemical polynucleotide synthesis methods are well known in the art and need not be described in detail herein. Those skilled in the art can use the sequences provided herein and commercial DNA synthesizers to generate the desired DNA sequence.

To prepare polynucleotides using recombinant methods, polynucleotides containing the desired sequence can be inserted into a suitable vector, and then the vector can be introduced into a suitable host cell for replication and amplification, as discussed further herein. The polynucleotide can be inserted into the host cell by any method known in the art. Cells are transformed by introducing exogenous polynucleotides by direct absorption, endocytosis, transfection, F-pairing, or electroporation. Once introduced, the exogenous polynucleotide can be maintained as a non-integrating vector (such as a plastid) in the cell or integrated into the host cell genome. The polynucleotide so amplified can be isolated from the host cell by methods well known in the art. See, for example, Sambrook et al., 1989.

Alternatively, PCR allows DNA sequence replication. PCR technology is well known in the art and described in US Patent Nos. 4,683,195, 4,800,159, 4,754,065, and 4,683,202, and PCR: The Polymerase Chain Reaction, edited by Mullis et al., Birkauswer Press, Boston, 1994.

RNA can be obtained by using isolated DNA in a suitable vector and inserting it into a suitable host cell. When cells replicate and DNA is transcribed into RNA, RNA can then be isolated using methods well known to those skilled in the art, for example, as described in Sambrook et al., 1989.

In some embodiments, the first vector comprises a polynucleotide encoding a heavy chain and the second vector comprises a polynucleotide encoding a light chain. In some embodiments, the first vector and the second vector are transfected into the host cell in a similar amount (such as a similar molar amount or similar quality). In some embodiments, the first vector and the second vector having a molar ratio or mass ratio between 5: 1 and 1: 5 are transfected into the host cell. In some embodiments, a vector encoding a heavy chain and a vector encoding a light chain with a mass ratio between 1: 1 and 1: 5 are used. In some specific examples, a vector encoding a heavy chain and a vector encoding a light chain with a mass ratio of 1: 2 are used.
Carrier

In some embodiments, a carrier optimized to express the polypeptide in CHO or CHO-derived cells or in NSO cells is selected. Exemplary such vectors are described in, for example, Running Deer et al., Biotechnol. Prog. 20: 880-889 (2004).

Suitable breeding and expression vectors can include various components, such as promoters, enhancers, and other transcriptional regulatory sequences. Vectors can also be constructed to allow subsequent colonization of antibody variable domains into different vectors. Suitable selection vectors can be constructed according to standard techniques, or can be selected from a large number of selection vectors available in this technique. Although the selected colony vector may vary according to the host cell intended, the suitable colony vector will generally be capable of self-replication, may have a single target with specific restriction endonuclease, and / or may carry a vector that can be used to select the containing vector Genes of pure line markers. Suitable examples include plastid and bacterial viruses such as pUC18, pUC19, Bluescript (eg pBS SK +) and its derivatives, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA and shuttle vectors such as pSA3 and pAT28. These and many other colonization vectors can be purchased from commercial suppliers such as BioRad, Strategene and Invitrogen. Further provide performance vectors. The expression vector is usually a replicable polynucleotide construct containing the polynucleotide according to the invention. It is implied that the expression vector must be replicable in the host cell as an episomal body or as an integral part of chromosomal DNA. Suitable expression vectors include, but are not limited to, plastids, viral vectors (including adenoviruses, adeno-associated viruses, retroviruses), cosmids, and expression vectors disclosed in PCT Publication No. WO 87/04462. Vector components may generally include (but are not limited to) one or more of the following: a signal sequence; an origin of replication; one or more marker genes; suitable transcription control elements (such as promoters, enhancers, and terminators). For performance (ie, translation), one or more translation control elements, such as ribosome binding sites, translation initiation sites, and stop codons are also usually required.

The vector containing the polynucleotide of interest and / or the polynucleotide itself can be introduced into the host cell by any of a variety of suitable methods, including electroporation, use of calcium chloride, rubidium chloride, calcium phosphate, DEAE -Transfection of polydextrose or other substances; microprojectile bombardment; liposome transfection; and infection (for example where the carrier is an infectious agent such as vaccinia virus). The choice of introducing a vector or polynucleotide will often depend on the characteristics of the host cell.
Host cell

Antibodies or antigen-binding fragments thereof can be produced recombinantly using a suitable host cell. Nucleic acids encoding antibodies or antigen-binding fragments can be cloned into expression vectors, which can then be introduced into host cells, such as E. coli cells, yeast cells, insect cells, monkey COS cells, Chinese hamster ovary (CHO) Cells or myeloma cells, where the cells do not additionally produce immunoglobulins to obtain the synthesis of antibodies in recombinant host cells. Preferred host cells include CHO cells, human fetal kidney HEK-293 cells or Sp2.0 cells, and various other cells well known in the art. Antibody fragments can be produced by proteolysis or other degradation of full-length antibodies, by recombinant methods, or by chemical synthesis. Polypeptide fragments of antibodies (especially shorter polypeptides of up to about 50 amino acids) can preferably be manufactured by chemical synthesis. Chemical synthesis methods for proteins and peptides are known in the art and are commercially available.

In various embodiments, the anti-CXCR5 heavy chain and / or anti-CXCR5 light chain may be expressed in prokaryotic cells, such as bacterial cells; or in eukaryotic cells, such as fungal cells (such as yeast), plant cells, insect cells, and Mammalian cells. Such performance can be performed, for example, according to procedures known in the art. Exemplary eukaryotic cells that can be used to express polypeptides include (but are not limited to) COS cells, including COS 7 cells; 293 cells, including 293-6E cells; CHO cells, including CHO-S, DG44. Lecl3 CHO cells, and FUT8 CHO cells ; PER.C6® cells (Crucell); and NSO cells. In some embodiments, the anti-CXCR5 heavy chain and / or anti-CXCR5 light chain may be expressed in yeast. See, for example, US Publication No. 2006/0270045 Al. In some embodiments, the particular eukaryotic host cell line is selected based on its ability to produce the desired post-translational modification against the CXCR5 heavy chain and / or against the CXCR5 light chain. For example, in some embodiments, CHO cells produce polypeptides that have higher levels of sialylation than the same polypeptide produced in 293 cells.

Introduction of one or more nucleic acids into the desired host cell can be accomplished by any method, including (but not limited to) calcium phosphate transfection, DEAE-polyglucose-mediated transfection, cationic lipid-mediated transfection, electroporation, transfection Lead, infection, etc. Non-limiting exemplary methods are described in, for example, Sambrook et al., Molecular Cloning, A Laboratory Manual, 3rd Edition Cold Spring Harbor Laboratory Press (2001). The nucleic acid can be transiently or stably transfected into the desired host cell according to any suitable method.

In some embodiments, the defucosylated anti-CXCR5 anti-system is prepared in cells capable of producing defucosylated antibodies, such as Lec3 CHO cells lacking protein fucosylation (Ripka et al. Arch. Biochem Biophys. 249: 533-545 (1986); US Patent Application No. US 2003/0157108; and WO 2004/056312, especially Example 11), and gene knockout cell lines, such as lack of functional α-1,6-rock Cell lines of trehalosyl transferase gene FUT8 , for example, knockout CHO cells (see, for example, Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda et al., Biotechnol. Bioeng, 94 (4): 680- 688 (2006); and WO2003 / 085107). In some embodiments, the defucosylated anti-CXCR5 anti-system is prepared in CHO cells lacking the functional FUT8 gene. In some embodiments, the defucosylated anti-CXCR5 anti-system is prepared in Potelligent® CHOK1SV cells (BioWa / Lonza, Allendale, NJ).

Anti-CXCR5 antibodies can be purified by any suitable method. Such methods include, but are not limited to, the use of affinity matrices or hydrophobic interaction chromatography. Suitable affinity ligands include CXCR5 ECD and ligands that bind to the constant region of the antibody. For example, protein A, protein G, protein A / G, or antibody affinity columns can be used to bind the constant region and purify anti-CXCR5 antibodies. Hydrophobic interaction chromatography (such as butyl or phenyl columns) is also suitable for the purification of some polypeptides. Many methods for purifying polypeptides are known in the art.

In some embodiments, the anti-CXCR5 antibody is produced in a cell-free system. Non-limiting exemplary cell-free systems are described in, for example, Sitaraman et al., Methods Mol. Biol. 498: 229-44 (2009); Spirin, Trends Biotechnol. 22: 538-45 (2004); Endo et al., Biotechnol. Adv . 21: 695-713 (2003).
V. Use and medical therapy

In some aspects, the invention provides therapeutic methods that use an anti-CXCR5 antibody or antigen-binding fragment thereof to remove, inhibit or reduce CXCR5 activity or reduce signaling, wherein the therapeutic methods include administering a therapeutically effective amount of an anti-CXCR5 antibody Or a pharmaceutical composition of an antigen-binding fragment thereof. The condition being treated is any disease or condition that can be improved, improved, inhibited or prevented by removing, inhibiting or reducing CXCR5 activity or reducing signaling.

In some aspects, the present invention provides anti-CXCR5 antibodies or antigen-binding fragments thereof for use in removing, inhibiting or reducing CXCR5 activity or reducing signaling. In some embodiments, the use may include administering a therapeutically effective amount of a pharmaceutical composition comprising an anti-CXCR5 antibody or antigen-binding fragment thereof. In some embodiments, the inhibition or reduction of CXCR5 activity or signaling can treat any disease or condition that is improved, improved, inhibited, or prevented by removing, inhibiting, or reducing CXCR5 activity or signaling.

Such diseases, disorders or conditions include, but are not limited to, inflammatory reactions, such as systemic lupus erythematosus (SLE); chronic inflammatory reactions; atherosclerosis; leukocyte adhesion deficiency; rheumatoid arthritis; diabetes (e.g. I Type 2 diabetes or insulin-dependent diabetes); multiple sclerosis; Raynaud's syndrome; autoimmune thyroiditis; allergic encephalomyelitis; Huguelin's syndrome; juvenile-onset diabetes; and usually tuberculosis, sarcoma , Polymyositis, granuloma and vasculitis, the immune response related to acute and delayed allergic reactions mediated by interleukins and T lymphocytes; Wegener's disease; pernicious anemia (Addison's Disease); diseases involving leukocyte exudation; inflammation of the central nervous system (CNS); multiple organ damage syndrome; hemolytic anemia (including but not limited to cryoglobulinemia or Qom's-positive anemia); myasthenia gravis; antigen -Antibody complex-mediated diseases; anti-glomerular basement membrane disease; anti-phospholipid syndrome; allergic neuritis; Graves' disease; Lambert-Eton muscle weakness syndrome; bullous pemphigoid; pemphigus; Autoimmune multiple endocrine lesions; white spot disease; Wright's disease; stiff syndrome; Behcet's disease; giant cell arteritis; immune complex nephritis; IgA nephropathy; IgM polyneuropathy; immune thrombocytopenic purpura (ITP) ) Or autoimmune thrombocytopenia and autoimmune hemolytic disease; Hashimoto's thyroiditis; autoimmune hepatitis; autoimmune hemophilia; autoimmune lymphoproliferative syndrome (ALPS); autoimmune uveitis retinitis ; Gerard-Barr's syndrome; Goodbold's syndrome; mixed connective tissue disease; autoimmune-related infertility; polyarteritis nodosa; alopecia areata; idiopathic mucus edema; graft-versus-host disease; muscle Atrophy (Duchen's type, Becker type, muscle tone type, limb band type, scapular arm type, congenital type, ophthalmopharyngeal type, distal type, Aide type) and cancer cells that control CXCR5 For example, the proliferation of pancreatic cancer, colon cancer, bladder cancer, T-cell leukemia, and B-cell leukemia is understood by those skilled in the art in consideration of the teachings disclosed herein.

The anti-CXCR5 antibody or antigen-binding fragment thereof of the present invention can also be used to detect and / or measure CXCR5 or CXCR5 expressing cells in a sample for diagnostic purposes, for example. For example, an anti-CXCR5 antibody or fragment thereof can be used to diagnose a condition or disease characterized by abnormal performance of CXCR5 (eg, over performance, low performance, lack of performance, etc.). An exemplary diagnostic analysis of CXCR5 may include, for example, contacting a sample obtained from a patient with an anti-CXCR5 antibody of the invention, where the anti-CXCR5 antibody is labeled with a detectable marker or reporter molecule.

As used herein, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" includes any material that, when combined with an active ingredient, allows the ingredient to retain biological activity and does not react with the individual's immune system. Examples include but are not limited to any of the carriers in standard medicine, such as phosphate buffered saline solutions, water, emulsions (such as oil / water emulsions), and various types of wetting agents. The preferred diluent for aerosol or parenteral administration is phosphate buffered saline (PBS) or physiological saline (0.9%). Compositions containing these carriers are formulated by well-known conventional methods (see, for example, Remington's Pharmaceutical Sciences, 18th edition, edited by A. Gennaro, Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy 20th edition, Mack Publishing, 2000).
VI . Composition

The present invention also provides a pharmaceutical composition comprising an effective amount of the CXCR5 antibody or antigen-binding fragment thereof described herein. Examples of these compositions and how to formulate these compositions are also described herein. In some embodiments, the composition includes one or more CXCR5 antibodies. In other embodiments, the CXCR5 antibody recognizes CXCR5. In other embodiments, the CXCR5 anti-systemic human antibody. In other embodiments, the CXCR5 anti-systemic humanized antibody. In some embodiments, the CXCR5 antibody contains a constant region capable of triggering a desired immune response, such as antibody-mediated lysis or ADCC. In other embodiments, the CXCR5 antibody comprises a constant region that is defucosylated and provides enhanced ADCC compared to antibodies that are otherwise identical in fucosylated. In other embodiments, the CXCR5 antibody comprises one or more CDRs of the antibody (such as one, two, three, four, five, or in some embodiments all six CDRs).

It should be understood that the composition may include more than one CXCR5 antibody or antigen-binding fragment thereof (eg, a mixture of CXCR5 antibodies that recognize different epitopes of CXCR5). Other exemplary compositions include more than one CXCR5 antibody that recognizes the same epitope of CXCR5 antibodies that bind to different epitopes of CXCR5, or different species. In some embodiments, the composition comprises a mixture of CXCR5 antibodies that recognize different variants of CXCR5.

The compositions used in the present invention may further comprise pharmaceutically acceptable carriers, excipients or stabilizers in the form of frozen formulations or aqueous solutions (Remington: The Science and practice of Pharmacy 20th Edition, 2000, Lippincott Williams and Wilkins, Ed. KE Hoover). Acceptable carriers, excipients or stabilizers are non-toxic to recipients at doses and concentrations, and may contain buffers such as phosphates, citrates and other organic acids; antioxidants, including ascorbic acid and methionine; Preservatives (such as octadecyldimethylbenzylammonium chloride; hexahydroxyammonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens , Such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) ) Polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, aspartame, histidine, Arginine or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or polydextrose; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; Salt-forming counterions, such as sodium; metal complexes (eg, Zn-proteins); and / or nonionic surfactants, such as TWEEN ™, PLURONICS ™, or polyethylene glycol (PEG). The pharmaceutically acceptable excipients will be further described herein.

The CXCR5 antibody, its antigen-binding fragment and its composition can also be used in combination with other reagents to enhance and / or supplement the effect of the reagent.

In certain embodiments, the CXCR5 antibody or antigen-binding fragment thereof is complexed with CXCR5 before administration. In certain embodiments, the CXCR5 antibody does not complex with CXCR5 before administration.

The present invention also provides a composition including any one of the pharmaceutical composition and the polynucleotide of the present invention. In some embodiments, the composition comprises an expression vector, which comprises a polynucleotide encoding an antibody as described herein. In other embodiments, the composition comprises a expression vector, which comprises a polynucleotide encoding any of the antibodies described herein. In still other embodiments, the composition includes each of the following: either or both of the polynucleotides encoding the sequences shown in SEQ ID NO: 1 and SEQ ID NO: 6; the code is shown in SEQ ID NO: 1 and either or both of the polynucleotides of the sequence in SEQ ID NO: 10; the codes in the polynucleotides of the sequences shown in SEQ ID NO: 1 and SEQ ID NO: 12 Either or both; either or both of the polynucleotides encoding the sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6; the encoding shown in SEQ ID NO: 5 and SEQ ID NO : Either or both of the polynucleotides of the sequence in 10; either or both of the polynucleotides that encode the sequences shown in SEQ ID NO: 5 and SEQ ID NO: 12; Either or both of the polynucleotides encoding the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 17; the polymers encoding the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 18 Either or both of the nucleotides; either or both of the polynucleotides encoding the sequences shown in SEQ ID NO: 13 and SEQ ID NO: 63; the encoding is shown in SEQ ID NO: 1 and either or both of the polynucleotides of the sequence in SEQ ID NO: 52; either of the polynucleotides encoding the sequences shown in SEQ ID NO: 1 and SEQ ID NO: 53 Or both; either or both of the polynucleotides encoding the sequences shown in SEQ ID NO: 1 and SEQ ID NO: 54; the codes shown in SEQ ID NO: 1 and SEQ ID NO: 55 Either or both of the polynucleotides of the sequence in the code; either or both of the polynucleotides of the sequence shown in SEQ ID NO: 1 and SEQ ID NO: 56; the code shows Either or both of the polynucleotides of the sequences in SEQ ID NO: 1 and SEQ ID NO: 57; the polynucleosides encoding the sequences shown in SEQ ID NO: 6 and SEQ ID NO: 48 Either or both of the acids; either or both of the polynucleotides encoding the sequences shown in SEQ ID NO: 6 and SEQ ID NO: 49; the encoding shown in SEQ ID NO: 6 and Either or both of the polynucleotides of the sequence in SEQ ID NO: 50; either of the polynucleotides encoding the sequences shown in SEQ ID NO: 6 and SEQ ID NO: 51 or Both; either or both of the polynucleotides encoding the sequences shown in SEQ ID NO: 17 and SEQ ID NO: 58; the codes shown in SEQ ID NO: 17 and S EQ ID NO: Either or both of the polynucleotides of the sequence in 59; either of the polynucleotides encoding the sequences shown in SEQ ID NO: 17 and SEQ ID NO: 60 or Both; either or both of the polynucleotides encoding the sequences shown in SEQ ID NO: 17 and SEQ ID NO: 61; the codes shown in SEQ ID NO: 17 and SEQ ID NO: 62 Either or both of the polynucleotides of the sequence; either or both of the polynucleotides encoding the sequence shown in SEQ ID NO: 52 and SEQ ID NO: 1; the encoding is shown in SEQ ID NO: 52 and either or both of the polynucleotides of the sequence in SEQ ID NO: 5; the coding is shown in SEQ ID NO: 52 and the polynucleotide of the sequence in SEQ ID NO: 47 Either or both; either or both of the polynucleotides encoding the sequences shown in SEQ ID NO: 52 and SEQ ID NO: 48; the encoding shown in SEQ ID NO: 52 and SEQ ID Either or both of the polynucleotides of the sequence in NO: 49; either or both of the polynucleotides that encode the sequences shown in SEQ ID NO: 52 and SEQ ID NO: 50 ; Either or both of the polynucleotides encoding the sequences shown in SEQ ID NO: 52 and SEQ ID NO: 51; or the sequences shown in SEQ ID NO: 35 and SEQ ID NO: 36 Either or both of the polynucleotides.

In another aspect, the polynucleotide may encode VH, VL, and / or both VH and VL of the antibody of the invention. That is, the composition comprises one polynucleotide or more than one polynucleotide encoding the antibody or antigen-binding fragment thereof of the present invention.

The pharmaceutical composition of the present invention can also be administered in combination therapy, such as in combination with other agents. For example, the combination therapy may include the CXCR5 antibody of the present invention or an antigen-binding fragment thereof in combination with at least one other therapy, where the therapy may be surgery, immunotherapy, or drug therapy.

The pharmaceutical compound of the present invention may include one or more pharmaceutically acceptable salts. Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids, such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic phosphorous acid, and the like; and those derived from non-toxic organic acids, such as Aliphatic monocarboxylic acid and aliphatic dicarboxylic acid, phenyl substituted alkanoic acid, hydroxyalkanoic acid, aromatic acid, aliphatic and aromatic sulfonic acid and the like. Base addition salts include those derived from alkaline earth metals (such as sodium, potassium, magnesium, calcium, and the like) and those derived from non-toxic organic amines (such as N, N′-benzylethylenediamine, N-methylglucose Amine, chloroprocaine (chloroprocaine), choline, diethanolamine, ethylenediamine, procaine (procaine and the like) salts.

The pharmaceutical composition of the present invention may also include a pharmaceutically acceptable antioxidant. Examples of pharmaceutically acceptable antioxidants include: (1) Water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) Oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanise ether (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol and the like; And (3) metal chelating agents such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid and the like.

Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical composition of the present invention include water, ethanol, polyols (such as glycerin, propylene glycol, polyethylene glycol and the like) and suitable mixtures thereof, vegetable oils (such as olive oil ) And injectable organic esters (such as ethyl oleate). Appropriate fluidity can be maintained, for example, by using coating materials such as lecithin, in the case of dispersions by maintaining the desired particle size, and by using surfactants.

These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. Preventing the presence of microorganisms can be ensured by sterilization procedures and by including various antibacterial and antifungal agents (for example, parabens, chlorobutanol, phenol, sorbic acid, and the like). It may also be desirable to include isotonic agents in the composition, such as sugar, sodium chloride and the like. In addition, prolonged absorption of injectable pharmaceutical forms can be achieved by including agents that delay absorption, such as aluminum monostearate and gelatin.

Pharmaceutical compositions generally must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable for higher drug concentration. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerin, propylene glycol, and liquid polyethylene glycol and the like) and suitable mixtures thereof. The proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the desired particle size in the case of dispersions, and by using surfactants. In most cases, it is suitable to include isotonic agents, for example, sugar, polyalcohols (such as mannitol, sorbitol), or sodium chloride in the composition. Prolonged absorption of injectable compositions can be achieved by including delayed absorption agents (such as monostearate and gelatin) in the composition.

Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microfiltration.

In general, dispersions are prepared by incorporating the active compound into a sterile vehicle containing an alkaline dispersion medium and the required other ingredients from those enumerated above. In the case where sterile powders are used to prepare sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization), which produces a powder of the active ingredient plus any additional desired ingredients from its previously sterile-filtered solution.

The pharmaceutical composition of the present invention may be suitable for preparation, encapsulation or sale of formulations administered via the eye. Such formulations may, for example, be in the form of eye drops in a solution or suspension including, for example, 0.1% -1.0% (w / w) active ingredient in an aqueous solution or an oily liquid carrier. Such drops may further include buffers, salts, or one or more other additional ingredients described herein. Other suitable formulations that can be administered by eye include those containing the active ingredient in the microcrystalline form or liposome preparation.

As used herein, "additional ingredients" include (but are not limited to) one or more of the following: excipients; surfactants; dispersants; inert diluents; granulating and disintegrating agents; binders; lubricants; Sweeteners; flavoring agents; colorants; preservatives; physiologically degradable compositions, such as gelatin; aqueous vehicles and solvents; oily vehicles and solvents; suspending agents; dispersants or wetting agents; emulsifiers, Demulcent; buffer; salt; thickener; filler; emulsifier; antioxidant; antibiotic; antifungal agent; stabilizer; and pharmaceutically acceptable polymeric or hydrophobic substances. Other "additional ingredients" that the pharmaceutical composition of the present invention may include are known in the art and described in, for example, Remington's Pharmaceutical Sciences, edited by Genaro, Mack Publishing Company, Easton, PA (1985), which is incorporated by reference In this article.

In one embodiment, the CXCR5 antibody or antigen-binding fragment thereof is administered in an intravenous formulation as a sterile aqueous solution containing 5 mg / mL, or in some embodiments about 10 mg / mL, or in some embodiments About 15 mg / mL in some cases, or about 20 mg / mL in some embodiments, or about 25 mg / mL in some embodiments, or about 50 mg / mL in some embodiments, and sodium acetate, Polysorbate 80 and sodium chloride with a pH in the range of about 5 to 6. In some embodiments, the intravenous formulation is a sterile aqueous solution containing 5 or 10 mg / mL antibody, and 20 mM sodium acetate, 0.2 mg / mL polysorbate 80, and 140 mM sodium chloride at pH 5.5 . In addition, solutions containing antibodies or antigen-binding fragments may contain histidine, mannitol, sucrose, trehalose, glycine, poly (ethylidene glycol), EDTA, methionine among many other compounds Acids and any combination thereof, as well as many other compounds known in the related art.

In one embodiment, the pharmaceutical composition of the present invention comprises the following components: 50 mg / mL CXCR5 antibody or antigen-binding fragment of the present invention, 20 mM histidine, 8.5% sucrose, and 0.02% polysorbate 80, 0.005 % EDTA at pH 5.8; in another embodiment, the pharmaceutical composition of the present invention comprises the following components: 100 mg / mL of the CXCR5 antibody or antigen-binding fragment of the present invention, 10 mM histidine, 5% sucrose, and 0.01 % Polysorbate 80 at pH 5.8. This composition can be provided in the form of a liquid formulation or lyophilized powder. When the powder is restored at full volume, the composition remains the same formula. Alternatively the powder can be reconstituted at half volume, in which case the composition contains 100 mg of the CXCR5 antibody of the invention or antigen-binding fragment thereof, 20 mM histidine, 10% sucrose and 0.02% polysorbate at pH 5.8醇 ester80.

In one embodiment, part of the dose is administered by intravenous administration and the rest is injected by antibody formulation. For example, 0.01 mg / kg of intravenously injected CXCR5 antibody or antigen-binding fragment thereof can be provided by a bolus, and the rest of the antibody dose can be administered by intravenous injection. The predetermined dose of CXCR5 antibody or antigen-binding fragment thereof can be administered, for example, over a period of one hour and half an hour to two hours to five hours.

With regard to therapeutic agents in which the agent is, for example, a small molecule, it may be present in the pharmaceutical composition in the form of a physiologically acceptable ester or salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.

The formulation of the pharmaceutical composition described herein can be prepared by any method known in the pharmacological technique or developed thereafter. Generally speaking, these preparation methods include the steps of combining the active ingredient with a carrier or one or more other accessory ingredients, and then shaping or encapsulating the product into the desired single-dose unit or multiple-dose unit as necessary or necessary .

In one embodiment, the composition of the present invention is a pyrogen-free formulation that is substantially free of endotoxin and / or related pyrolytic substances. Endotoxins include toxins that are restricted to microorganisms and that are released when the microorganisms break down or die. Pyrolysis substances also include pyrogenic and thermally stable substances (glycoproteins) from the outer membrane of bacteria and other microorganisms. If administered to humans, both of these substances can cause fever, hypotension and shock. Due to the potential harmful effects, it is advantageous to remove even low amounts of endotoxin from intravenous administration of pharmaceutical drug solutions. The Food and Drug Administration ("FDA") has set an upper limit of 5 endotoxin units (EU) for intravenous drug application per dose per kilogram of body weight within one hour (The United States Pharmacopeial Convention, Pharmacopeial Forum 26 (1) : 223 (2000)). When the therapeutic protein is administered in an amount of several hundred milligrams or several thousand milligrams per kilogram of body weight, even removing trace amounts of endotoxin is advantageous. In one embodiment, the endotoxin and pyrogen content in the composition is less than 10 EU / mg, or less than 5 EU / mg, or less than 1 EU / mg, or less than 0.1 EU / mg, or less than 0.01 EU / mg , Or less than 0.001 EU / mg. In another embodiment, the endotoxin and pyrogen content in the composition is less than about 10 EU / mg, or less than about 5 EU / mg or, less than about 1 EU / mg, or less than about 0.1 EU / mg, or Less than about 0.01 EU / mg, or less than about 0.001 EU / mg.

In one embodiment, the invention comprises an administration composition, wherein the administration is oral, parenteral, intramuscular, intranasal, vaginal, transrectal, translingual, sublingual, transbuccal, intrabuccal, intravenous , Skin, subcutaneous or transdermal administration.

In another embodiment, the present invention further comprises an administration composition, which is combined with other treatments, such as surgery, chemotherapy, hormone therapy, biological therapy, immunotherapy or radiation therapy.
VII. Administration / administration

To prepare a pharmaceutical or sterile composition including the CXCR5 antibody or antigen-binding fragment of the present invention, these antibodies are mixed with a pharmaceutically acceptable carrier or excipient. The formulations of therapeutic agents and diagnostic agents can be formulated with physiologically acceptable carriers, excipients, or stabilizers in the form of, for example, lyophilized powders, slurries, aqueous solutions, lotions, or suspensions (see, for example, Hardman , Et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams and Wilkins, New York, NY; Avis, etc. People (ed.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman et al. (Ed.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman et al. (Ed.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY).

The choice of administration regimen for treatment depends on several factors, including the serum or tissue turnover rate of the entity, the degree of symptoms, the immunogenicity of the entity, and the accessibility of the target cells in the biological matrix. In certain embodiments, the dosing regimen maximizes the therapeutic dose delivered to the patient based on acceptable levels of side effects. Therefore, the amount of biological agent delivered depends in part on the specific entity and the severity of the condition being treated. Guidance for selecting appropriate doses of antibodies, cytokines and small molecules can be obtained (see, for example, Wawrzynczak, 1996, Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.), 1991, Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY; Bach (eds.), 1993, Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY; Baert et al., 2003, New Engl. J. Med. 348: 601-608 ; Milgrom et al., 1999, New Engl. J. Med. 341: 1966-1973; Slamon et al., 2001, New Engl. J. Med. 344: 783-792; Beniaminovitz et al., 2000, New Engl. J. Med. 342: 613-619; Ghosh et al., 2003, New Engl. J. Med. 348: 24-32; Lipsky et al., 2000, New Engl. J. Med. 343: 1594-1602).

Appropriate dosages are determined by clinicians, for example, using parameters or factors known or suspected in the art that affect or predict treatment. In general, the initial dose is an amount that is slightly less than the optimal dose, and then is increased in small increments until the desired or optimal effect (relative to any negative side effects) is achieved. Important diagnostic measurements include, for example, symptoms of inflammation or the level of inflammatory cytokines produced.

The actual dosage concentration of the active ingredient in the pharmaceutical composition of the present invention can be changed in order to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration without toxicity to the patient. The selected dose will depend on various pharmacokinetic factors, including the activity of the specific composition used in the present invention or its ester, salt or amide; the route of administration; the time of administration; the excretion rate of the specific compound used; the duration of treatment; and the use Other drugs, compounds, and / or substances used in combination with a particular composition; the age, sex, weight, condition, general health status, and previous medical history of the patient being treated; and similar factors well known in medical technology.

The composition comprising the CXCR5 antibody or antigen-binding fragment thereof of the present invention can be provided by continuous infusion, for example, by administration at intervals of 1 to 7 times a day, week, or week. The dose may be given intravenously, subcutaneously, superficially, orally, nasally, rectally, intramuscularly, intracranially or by inhalation. Specific dose agreements are agreements that involve a maximum dose or dose frequency that avoids a large number of undesirable side effects. The total weekly dose may be at least 0.05 μg / kg body weight, at least 0.2 μg / kg, at least 0.5 μg / kg, at least 1 μg / kg, at least 10 μg / kg, at least 100 μg / kg, at least 0.2 mg / kg, at least 1.0 mg / kg, at least 2.0 mg / kg, at least 10 mg / kg, at least 15 mg / kg, at least 20 mg / kg, at least 25 mg / kg or at least 50 mg / kg (see for example Yang, et al., 2003, New Engl. J. Med. 349: 427-434; Herold, et al., 2002, New Engl. J. Med. 346: 1692-1698; Liu, et al., 1999, J. Neurol. Neurosurg. Psych. 67: 451 -456; Portielji, et al., 2003, Cancer. Immunol. Immunother. 52: 133-144). The dose may be at least 15 μg, at least 20 μg, at least 25 μg, at least 30 μg, at least 35 μg, at least 40 μg, at least 45 μg, at least 50 μg, at least 55 μg, at least 60 μg, at least 65 μg, at least 70 μg , At least 75 μg, at least 80 μg, at least 85 μg, at least 90 μg, at least 95 μg, or at least 100 μg. The dose administered to an individual may total at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 times, or more than 12 times.

For the CXCR5 antibody or antigen-binding fragment thereof of the present invention, the dose administered to the patient may be 0.0001 mg / kg to 100 mg / kg of the patient's body weight. The dosage can be between 0.0001 mg / kg and 20 mg / kg, 0.0001 mg / kg and 10 mg / kg, 0.0001 mg / kg and 5 mg / kg, 0.0001 and 2 mg / kg, 0.0001 and 1 mg / kg, 0.0001 mg / kg and 0.75 mg / kg, 0.0001 mg / kg and 0.5 mg / kg, 0.0001 mg / kg to 0.25 mg / kg, 0.0001 to 0.15 mg / kg, 0.0001 to 0.10 mg / kg, 0.001 to 0.5 mg / kg, 0.01 to Between 0.25 mg / kg or 0.01 to 0.10 mg / kg of the patient's body weight.

The dose of the CXCR5 antibody or antigen-binding fragment thereof can be calculated by multiplying the patient's weight in kilograms (kg) by the dose to be administered in mg / kg. The dose of the antibody of the present invention may be 150 μg / kg or less, 125 μg / kg or less, 100 μg / kg or less, 95 μg / kg or less, 90 μg / kg or less of the patient's body weight, 85 μ / kg or less, 80 μ / kg or less, 75 μ / kg or less, 70 μ / kg or less, 65 μ / kg or less, 60 μ / kg or less, 55 μ / kg or less, 50 μ / kg or less, 45 μ / kg or less, 40 μ / kg or less, 35 μ / kg or less, 30 μ / kg or less, 25 μ / kg Or less, 20 μ / kg or less, 15 μ / kg or less, 10 μ / kg or less, 5 μ / kg or less, 2.5 μ / kg or less, 2 μ / kg or less Small, 1.5 μ / kg or less, 1 μ / kg or less, 0.5 μ / kg or less, or 0.1 μ / kg or less.

The unit dose of the CXCR5 antibody or antigen-binding fragment of the present invention may be 0.1 mg to 200 mg, 0.1 mg to 175 mg, 0.1 mg to 150 mg, 0.1 mg to 125 mg, 0.1 mg to 100 mg, 0.1 mg to 75 mg, 0.1 mg to 50 mg, 0.1 mg to 30 mg, 0.1 mg to 20 mg, 0.1 mg to 15 mg, 0.1 mg to 12 mg, 0.1 mg to 10 mg, 0.1 mg to 8 mg, 0.1 mg to 7 mg, 0.1 mg To 5 mg, 0.1 mg to 2.5 mg, 0.25 mg to 20 mg, 0.25 mg to 15 mg, 0.25 mg to 12 mg, 0.25 mg to 10 mg, 0.25 mg to 8 mg, 0.25 mg to 7 mg, 0.25 mg to 5 mg, 0.5 mg to 2.5 mg, 1 mg to 20 mg, 1 mg to 15 mg, 1 mg to 12 mg, 1 mg to 10 mg, 1 mg to 8 mg, 1 mg to 7 mg, 1 mg to 5 mg, Or 1 mg to 2.5 mg.

The dose of the CXCR5 antibody or antigen-binding fragment thereof of the present invention can reach at least 0.1 μg / mL, at least 0.5 μg / mL, at least 1 μg / mL, at least 2 μg / mL, at least 5 μg / mL, at least 6 μg in an individual / mL, at least 10 μg / mL, at least 15 μg / mL, at least 20 μg / mL, at least 25 μg / mL, at least 50 μg / mL, at least 100 μg / mL, at least 125 μg / mL, at least 150 μg / mL , At least 175 μg / mL, at least 200 μg / mL, at least 225 μg / mL, at least 250 μg / mL, at least 275 μg / mL, at least 300 μg / mL, at least 325 μg / mL, at least 350 μg / mL, at least Serum titer of 375 μg / mL, or at least 400 μg / mL. Alternatively, the antibody dose of the present invention can reach at least 0.1 μg / ml, at least 0.5 μg / ml, at least 1 μg / ml, at least 2 μg / ml, at least 5 μg / ml, at least 6 μg / ml, At least 10 μg / ml, at least 15 μg / ml, at least 20 μg / ml, at least 25 μg / ml, at least 50 μg / ml, at least 100 μg / ml, at least 125 μg / ml, at least 150 μg / ml, at least 175 μg / ml, at least 200 μg / ml, at least 225 μg / ml, at least 250 μg / ml, at least 275 μg / ml, at least 300 μg / ml, at least 325 μg / ml, at least 350 μg / ml, at least 375 μg / ml Serum titer of ml or at least 400 μg / ml.

The CXCR5 antibody or antigen-binding fragment thereof of the present invention can be repeatedly administered and the administration can be at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or at least 6 months.

The effective amount for a particular patient can vary depending on factors such as the condition being treated, the overall health of the patient, the route of administration and the dosage and the severity of side effects (see, for example, Maynard, et al., 1996, A Handbook of SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, FIa .; Dent, 2001, Good Laboratory and Good Clinical Practice, Urch Publ, London, UK).

The route of administration can be, for example, by topical or cutaneous application, intravenously, intraperitoneally, intracerebrally, intramuscularly, intraocularly, intraarterially, intracerebrospinally, intralesionally or by infusion or by sustained release system or implant See, for example, Sidman et al., 1983, Biopolymers 22: 547-556; Langer, et al., 1981, J. Biomed. Mater. Res. 15: 167-277; Langer, 1982, Chem. Tech. 12: 98-105; Epstein, et al., 1985, Proc. Natl. Acad. Sci. USA 82: 3688-3692; Hwang, et al., 1980, Proc. Natl. Acad. Sci. USA 77: 4030-4034; US Patent No. 6,350466 And No. 6,316,024). If necessary, the composition may also include a solubilizer and a local anesthetic such as lidocaine to reduce pain at the injection site. In addition, administration via the lung may be used, for example, inhalers or nebulizers and formulations with aerosols. See, for example, U.S. Patent Nos. 6,019,968, 5,985,320, 5,985,309, 5,934,272, 5,874,064, 5,855,913, 5,290,540, and 4,880,078; and PCT Publication Nos. WO 92/19244, WO 97 / 32572, WO 97/44013, WO 98/31346, and WO 99/66903, each of which is incorporated herein by reference in its entirety. In one embodiment, Alkermes AIR ™ pulmonary drug delivery technology (Alkermes, Inc., Cambridge, Mass.) Is used to administer the CXCR5 antibody or antigen-binding fragment or composition of the invention.

The composition of the present invention can also be administered through one or more administration routes using one or more of various methods known in the art. As those skilled in the art understand, the route and / or mode of administration will vary depending on the desired result. Selected routes of administration for the antibodies of the invention include intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal cord, or other parenteral routes of administration, such as by injection or infusion. Non-enteric administration can mean the administration mode usually by injection except enteral and local administration, and includes (but not limited to) intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac , Intradermal, intraperitoneal, transtracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspine, epidural, and intrasternal injection and infusion. Alternatively, the compositions of the present invention can be administered via non-parenteral routes, such as topical, epidermal, or transmucosal routes, such as intranasal, oral, transvaginal, transrectal, sublingual, or topical.

If the CXCR5 antibody or antigen-binding fragment of the invention is administered in a controlled release or sustained release system, the pump can be used to achieve controlled or sustained release (see Langer, aforementioned; Sefton, 1987, CRC Crit. Ref. Biomed. Eng 14:20; Buchwald et al., 1980, Surgery 88: 501; Saudek et al., 1989, N. Engl. J. Med. 321: 514).

Polymeric materials can be used to achieve the controlled or sustained release of therapeutics invented (see, for example, Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Press, Boca Raton, FIa. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, 1983, J., Macromol. ScL Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science 11 225: 190; During et al., 19Z9, Ann. Neurol. 25: 351; Howard et al. 1989, J. Neurosurg. 71: 105); US Patent No. 5,679,377; US Patent No. 5,916,597; US Patent No. 5,912,015 No .; US Patent No. 5,989,463; US Patent No. 5,128,326; PCT Publication No. WO 99/15154; and PCT Publication No. WO 99/20253. Examples of polymers used in sustained release formulations include (but are not limited to) poly (2-hydroxyethyl methacrylate), poly (methyl methacrylate), poly (acrylic acid), poly (ethylene-co-acetic acid Vinyl ester), poly (methacrylic acid), polyglycolide (PLG), polyanhydride, poly (N-vinylpyrrolidone), poly (vinyl alcohol), polypropylene amide, poly (ethylene glycol), Polylactide (PLA), poly (lactide-co-glycolide) (PLGA) and polyorthoesters. In one embodiment, the polymer used in the sustained release formulation is inert, free of leachable impurities, stable in storage, sterile and biodegradable. Controlled or sustained release systems can be placed close to preventive or therapeutic targets, so only the part of the systemic dose is needed (see, for example, Goodson, in Medical Applications of Controlled Release, aforementioned, Volume 2, pages 115-138 (1984)) .

Controlled release systems are discussed in Langer, 1990, Science 249: 1527-1533. Any technique known to those skilled in the art can be used to produce sustained release formulations comprising one or more antibodies or conjugates of the invention. See, for example, US Patent No. 4,526,938; International Patent Publication Nos. WO 91/05548, WO 96/20698; Ning et al., 1996, "Intratumoral Radioimmunotheraphy of a Human Colon Cancer Xenograft Using a Sustained-Release Gel", Radiotherapy and Oncology 59: 179-189; Song et al., 1995, "Antibody Mediated Lung Targeting of Long-Circulating Emulsions", PDA Journal of Pharmaceutical Science and Technology 50: 372-397; Cleek et al., 1997, "Biodegradable Polymeric Carriers for a bFGF Antibody for Cardiovascular Application ", Pro. Ml. Symp. Control. Rel. Bioact. Mater. 24: 853-854; and Lam et al., 1997," Microencapsulation of Recombinant Humanized Monoclonal Antibody for Local Delivery ", Proc. Ml. Symp. Control Rel. Bioact. Mater. 24: 759-160, each of which is incorporated by reference in its entirety.

If the CXCR5 antibody or antigen-binding fragment of the invention is administered locally, it can be formulated into ointments, creams, transdermal patches, lotions, gels, shampoos, sprays, aerosols, solutions, emulsions Or other forms well known to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences and Introduction to Pharmaceutical Dosage Forms, 19th Edition, Mack Pub. Co., Easton, Pa. (1995). For non-sprayable topical dosage forms, a viscous-semi-solid or solid form containing a carrier or one or more excipients compatible with topical application and in some cases having a dynamic viscosity greater than water is generally used. Suitable formulations include (but are not limited to) solutions, suspensions, emulsions, creams, ointments, powders, wipes, ointments, and the like, which are sterilized or supplemented with auxiliary agents (eg, preservatives, stabilizers, Wetting agents, buffers or salts) are mixed to affect various characteristics, such as osmotic pressure. Other suitable topical dosage forms include sprayable aerosol formulations, where, in some cases, the active ingredient combined with a solid or liquid inert carrier is encapsulated in a mixture with pressurized volatiles (eg, gaseous propellants, such as freon) Or squeeze the bottle. If necessary, a humectant or humectant can also be added to the pharmaceutical composition and dosage form. Examples of such other ingredients are well known in the art.

If the composition containing the CXCR5 antibody or antigen-binding fragment thereof is administered intranasally, it can be formulated into an aerosol form, spray, mist or drop form. In particular, the prophylactic or therapeutic agent used in accordance with the present invention may be preferably delivered in the form of an aerosol spray presented from a pressurized pack or nebulizer, in which a suitable propellant (eg Chlorotetrafluoroethane, carbon dioxide or other suitable gases). In the case of pressurized aerosols, the dosage unit can be determined by providing a valve that delivers a metered quantity. Capsules and cartridges (made of, for example, gelatin) containing compound powder mixtures of the compound and a suitable powder base such as lactose or starch can be formulated for use in inhalers or insufflators.

Methods for co-administration or treatment with a second therapeutic agent (eg, cytokines, steroids, chemotherapeutics, antibiotics, or radiation) are well known in the art (see, for example, Hardman et al. (Eds.) (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th edition, McGraw-Hill, New York, NY; Poole and Peterson (ed.) (2001) Pharmacotherapeutics for Advanced Practice: A Practical Approach, Lippincott, Williams and Wilkins, Phila., Pa .; Chabner and Longo (ed.) (2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams and Wilkins, Phila., Pa.). An effective amount of the therapeutic agent can reduce symptoms by at least 10%; at least 20%; at least about 30%; at least 40% or at least 50%.

It can be administered in combination with the CXCR5 antibody or antigen-binding fragment of the present invention for additional therapies (eg, prophylactic or therapeutic agents) can be administered with the antibody of the present invention less than 5 minutes apart, less than 30 minutes apart, 1 hour apart, separated About 1 hour, about 1 to about 2 hours apart, about 2 hours to about 3 hours apart, about 3 hours to about 4 hours apart, about 4 hours to about 5 hours apart, about 5 hours to about 6 hours apart, about 6 hours to about 7 hours, about 7 hours to about 8 hours apart, about 8 hours to about 9 hours apart, about 9 hours to about 10 hours apart, about 10 hours to about 11 hours apart, about 11 hours to about 12 hours apart Hours, about 12 hours to 18 hours apart, 18 hours to 24 hours apart, 24 hours to 36 hours apart, 36 hours to 48 hours apart, 48 hours to 52 hours apart, 52 hours to 60 hours apart, 60 hours to 72 hours apart Hours, 72 to 84 hours apart, 84 to 96 hours apart, or 96 to 120 hours apart. Two or more therapies can be administered within the same patient consultation.

The CXCR5 antibody or antigen-binding fragment of the present invention and other therapeutic agents can be administered via circulation. Circulating therapy involves the administration of a first therapy (eg, a first prophylactic or therapeutic agent) for a period of time, followed by a second therapy (eg, a second prophylactic or therapeutic agent) for a period of time, and then a third therapy as appropriate (Eg, prophylactic or therapeutic) for a period of time, etc., and repeat this sequential administration (ie, circulation) in order to reduce resistance to one of the therapies, thereby avoiding or reducing the one of the therapies Side effects, and / or the efficacy of improved therapy.

In one embodiment, the CXCR5 antibody of the present invention can be co-administered with compositions for the treatment of autoimmune diseases and disorders, such compositions include but are not limited to adriamycin, azathioprine, Busulfan, cyclophosphamide, cyclosporine A, cyclophosphazide, fludarabine, 5-fluorouracil, methotrexate, mycophenolate mofetil, 6- Mercaptopurine, corticosteroids, non-steroidal anti-inflammatory agents, sirolimus (rapamycin) and tacrolimus (FK-506). In alternative embodiments, the immunomodulatory or immunosuppressive agent is an antibody selected from the group consisting of: moromona-CD3 (muromonab-CD3), alemtuzumab (Campath®), baliximab (basiliximab) ), Daclizumab, muromonab (OKT3®), rituximab, antithymocyte globulin and IVIg, and other antibodies known to those skilled in the art.

In one embodiment, the CXCR5 antibody of the present invention can be co-administered with a composition for the treatment of diabetes, such compositions include, but are not limited to, biguanide (eg, metformin, metformin, and phenformin), hormones, and the like (Amyloid, insulin, insulin aspart, insulin detemir, insulin glargine, insulin lysin, insulin lispro, liraglutide and pramlintide), sulfonylurea derivatives (acesulfame Cyclohexamide, Sulbutacarb, Chlorpropamide, Glyburide, Gliclazide, Glimepiride, Glipizide, Gliclone, Glipide, Glyburide, Glyburide Glipizole, Glipizole, Glipizide, Glipizide, Methazep, Uprofen, Tolbutamide, and Toluene Urea), Thiazolidinedione (Pioglitazone, Rosiglitazone, and Troglitazone) ), Acarbose, exenatide, miglitol, miglinide, mitiglinide, muraglitazar, nateglinide, repaglide Chennai (repaglinide), sitagliptin, sitaglipa, tesaglitazar, vildagliptin and voglibose.

In certain embodiments, the CXCR5 antibody or antigen-binding fragment of the invention can be formulated for proper distribution in vivo. For example, the blood-brain barrier (BBB) does not include many highly hydrophilic compounds. To ensure that the therapeutic agent of the present invention crosses BBB (if necessary), it can be formulated, for example, in liposomes. For methods of manufacturing liposomes, see, for example, US Patent Nos. 4,522,811; 5,374,548; and 5,399,331. Liposomes can contain one or more parts that are selectively delivered to specific cells or organs, thus enhancing target drug delivery (see, for example, V.V. Ranade, 1989, J. Clin. Pharmacol. 29: 685). Exemplary target parts include folic acid or biotin (see, eg, US Patent 5,416,016); mannose (Umezawa et al., Biochem. Biophys. Res. Commun. 153: 1038); antibodies (PG Bloeman et al., 1995, FEBS Lett 357: 140; M. Owais et al., 1995, Antimicrob. Agents Chemother. 39: 180); surfactant protein A receptor (Briscoe et al. (1995) Am. J. Physiol. 1233: 134); pl20 (Schreier et al. (1994) J. Biol. Chem. 269: 9090); see also K. Keinanen; ML Laukkanen, 1994, FEBS Lett. 346: 123; Killion; Fidler, 1994; Immunomethods 4: 273.

The present invention provides a solution for administering a pharmaceutical composition to an individual in need, the pharmaceutical composition comprising the CXCR5 antibody of the present invention alone or an antigen-binding fragment thereof, or a combination thereof with other therapies. The therapies (eg, prophylactic or therapeutic agents) of the combination therapy of the present invention can be administered to an individual simultaneously or continuously. The therapies (eg, prophylactic or therapeutic agents) of the combination therapy of the present invention can also be administered cyclically. Circulating therapy involves administration of the first therapy (eg, the first prophylactic or therapeutic agent) over a period of time, followed by administration of the second therapy (eg, the second prophylactic or therapeutic agent) over a period of time, and repeated sequential administration of the drug (i.e. Circulation) in order to reduce the development of resistance to one of the therapies (eg reagents) to avoid or reduce the side effects of one of the therapies (eg reagents) and / or to enhance the efficacy of the therapy.

The therapies (e.g., prophylactic or therapeutic agents) of the combination therapy of the present invention can be administered to an individual simultaneously. The term "simultaneously" is not limited to administration of therapies (such as prophylactic or therapeutic agents) at exactly the same time, but actually means that the pharmaceutical composition containing the CXCR5 antibody or antigen-binding fragment of the present invention is delivered to the individual sequentially and within a time interval Administration allows the antibody of the invention or its conjugate to work with other therapies to provide increased benefits compared to other methods. For example, the therapies can be administered to an individual simultaneously or in any order at different points in time; however, if they are not administered simultaneously, they should be administered when the time is sufficiently close to provide the desired therapeutic or preventive effect. Each therapy can be administered to the individual separately in any suitable form and by any suitable route. In various embodiments, it may be less than 15 minutes, less than 30 minutes, less than 1 hour, about 1 hour, about 1 hour to about 2 hours apart, about 2 hours to about 3 hours apart, about 3 hours to about 4 hours apart , About 4 hours to about 5 hours apart, about 5 hours to about 6 hours apart, about 6 hours to about 7 hours apart, about 7 hours to about 8 hours apart, about 8 hours to about 9 hours apart, about 9 hours apart Therapies (e.g., prophylactic or therapeutic) are administered to an individual up to about 10 hours, about 10 hours to about 11 hours apart, about 11 hours to about 12 hours apart, 24 hours apart, 48 hours apart, 72 hours apart, or 1 week apart Agent). In other embodiments, two or more therapies (eg, prophylactic or therapeutic agents) can be administered within the same patient consultation.

The prophylactic or therapeutic agent of combination therapy can be administered to an individual with the same pharmaceutical composition. Alternatively, the prophylactic or therapeutic agent of combination therapy can be administered to the individual simultaneously in a separate pharmaceutical composition. The prophylactic or therapeutic agent can be administered to the individual by the same or different administration route.
VIII . Set

The invention also provides kits comprising any or all of the antibodies described herein. The kit of the invention includes one or more containers containing the CXCR5 antibody described herein and instructions for use according to a method of the invention described herein. In general, these instructions include instructions for the administration of antibodies for the above therapeutic treatments. In some embodiments, kits are provided for generating single-dose dosing units. In some embodiments, the kit may contain a first container with dried protein and a second container with an aqueous formulation. In certain embodiments, kits containing applicators (eg, single-chamber and multi-chamber pre-filled syringes (eg, liquid syringes and freeze-dried syringes) are included.

Instructions related to the use of CXCR5 antibodies usually include information about the dosage, timing and route of administration for the intended treatment. The container may be in unit dose, bulk (eg multi-dose packaging) or sub-unit dose. The instructions supplied in the kit of the present invention are written instructions usually on the label or the instructions of the medicine (such as the paper included in the kit), but machine-readable instructions (such as the instructions on the magnetized or optical storage disk) are also acceptable Accepted.

The set of the present invention is in a suitable packaging form. Suitable packaging includes, but is not limited to, vials, bottles, cans, flexible packaging (such as sealed Mylar or plastic bags), and the like. Packaging for combination with a specific device, such as an inhaler, a nasal device (eg, a nebulizer), or an infusion device (such as a small pump) is also covered. The kit may have a sterile access hole (for example, the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic injection needle). The container may also have a sterile access hole (for example, the container may be an intravenous solution bag or a vial with a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is the CXCR5 antibody of the invention. The container may further contain a second pharmaceutically active agent.

The kit can provide additional components such as buffers and explanatory information as appropriate. Typically, the kit includes the container and a label or package insert on or associated with the container.

The invention also provides a diagnostic kit comprising any or all of the antibodies described herein. The diagnostic kit is suitable for detecting the presence of CXCR5 in samples, for example. In some embodiments, the diagnostic kit can be used to identify individuals with underlying diseases, disorders, or conditions that place the individual in a CXCR5-mediated disease, disorder, or condition, or CXCR5 deficient disease , Risk of illness or condition. In some embodiments, the diagnostic kit can be used to detect the presence and / or level of CXCR5 in an individual suspected of having a CXCR5-mediated disease or a CXCR5-deficient disease, disorder, or condition.

The diagnostic kit of the present invention includes one or more containers containing the CXCR5 antibody described herein and instructions for use according to a method of the present invention described herein. In general, these instructions include the use of CXCR5 antibodies to detect individuals at risk of or suspected of having a CXCR5-mediated disease or CXCR5-deficient disease, disorder or condition. Description of the existence of CXCR5 in In some embodiments, an exemplary diagnostic kit can be configured to contain reagents (such as CXCR5 antibodies, negative control samples, positive control samples) and instructions for using the kit.
IX . Equivalent

The foregoing description and the following examples detail certain specific embodiments of the invention and describe the best mode contemplated by the inventors. However, it will be understood that the present invention can be practiced in many ways, no matter how detailed the foregoing content presented in text, and the present invention should be interpreted in accordance with the scope of the attached patent application and any equivalents thereof.

Although the teachings disclosed have been described with reference to different applications, methods, kits, and composition descriptions, it will be understood that various changes and modifications can be made without departing from the teachings herein and the invention claimed below. The following examples are provided to better illustrate the teaching content of the present invention and are not intended to limit the scope of the teaching content presented herein. Although the teachings of the present invention have been described in terms of these exemplary embodiments, skilled artisans will readily understand that many variations and modifications of these exemplary embodiments are possible without undue experimentation. All such changes and modifications are within the scope of the current teaching.

All references cited in this article (including patent cases, patent applications, papers, textbooks, and the like) and references cited therein to the extent that they have not yet been cited, are hereby incorporated by reference in their entirety. In the event that one or more of the incorporated documents and similar materials (including but not limited to defined terms, term usage, described technology, or the like) is different or inconsistent with this application, the application is regarded as quasi.
X. General technology

It should be understood that the present invention is not limited to a specific synthetic preparation method, which may of course vary. Unless otherwise defined herein, the scientific and technical terms used in conjunction with the present invention shall have the meaning generally understood by those of ordinary skill. In addition, unless the context requires otherwise, singular terms shall include pluralities and plural terms shall include the singular. In general, the nomenclature and techniques described in this document that are used in conjunction with cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization are well known and commonly used in this technology By.

Unless otherwise specified, the practice of the present invention will employ conventional techniques in molecular biology (including recombinant technology), microbiology, cell biology, biochemistry, and immunology within this technology. These techniques are fully explained in documents such as: Molecular Cloning: A Laboratory Manual, Second Edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (edition by MJ Gait, 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (edited by JE Cellis, 1998) Academic Press; Animal Cell Culture (edited by RI Freshney, 1987); Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (edited by A. Doyle, JB Griffiths and DG Newell, 1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (edited by DM Weir and CC Blackwell); Gene Transfer Vectors for Mammalian Cells (Edited by JM Miller and MP Calos, 1987); Current Protocols in Molecular Biology (FM Ausubel et al., Edited, 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., Edited, 1994); Current Protocols in Immunology (JE Coligan et al., Editor, 1991); Sambrook and Russell, Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (20 01); Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, NY (2002); Harlow and Lane Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1998); Coligan et al Human, Short Protocols in Protein Science, John Wiley & Sons, NY (2003); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997 ); Antibodies: a practical approach (Edited by D. Catty., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, edited, Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and JD Capra, editor, Harwood Academic Publishers, 1995).

The enzymatic reaction and purification techniques are performed according to the manufacturer's instructions for such techniques or as described herein. The nomenclature used in conjunction with analytical chemistry, biochemistry, immunology, molecular biology, synthetic organic chemistry, and medical and medicinal chemistry, and their laboratory procedures and techniques described herein are well known and commonly used in this technology. Standard techniques are used in chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and patient treatment.
XI . Biological Hosting

The representative substance of the present invention was deposited at the American Type Culture Collection (American Type Culture Collection) on 10801 University Avenue, Manassas, Virginia, USA, on July 26, 2017. Vector h11G2-VH (XC155) with ATCC accession number PTA-124323 contains a DNA insert encoding the heavy chain variable region of antibody h11G2 (XC155), and vector h11G2-VL (XC154) with ATCC accession number PTA-124324 contains DNA insert encoding the light chain variable region of antibody h11G2 (XC154). Deposit in accordance with the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure (Budapest Treaty) and the regulations under it. This guarantee maintains the vitality cultivation of the deposit for 30 years from the date of deposit. Deposit will be provided by the ATCC under the terms of the Budapest Treaty, and is subject to the agreement between Pfizer Inc. and ATCC, which guarantees the publication of the relevant U.S. patent or the publication of any U.S. or foreign patent application to the public (in any order) , The public can permanently and unrestrictedly use the progeny of the deposit culture and is guaranteed by the U.S. Patent and Trademark Office members in accordance with 35 USC section 122 and in accordance with its committee rules (including specific reference 886 OG 638 of 37 CFR section 1.14) Those determined by the authorization can use the offspring.

The owner of this application has agreed that if the culture of the deposited substance dies, is lost or destroyed when cultivated under suitable conditions, it will notify to immediately replace the substance with another identical substance. The availability of deposited materials is not interpreted as a license to practice the invention in violation of the rights granted by any government department under its patent laws.
Examples

The present invention is described in further detail with reference to the following experimental examples. Unless otherwise specified, these examples are provided for illustrative purposes only and are not intended to be limiting. Therefore, the present invention should in no way be interpreted as being limited to the following examples, but should be interpreted to cover any and all variations that become apparent as a result of the teachings provided herein.
Example 1: Hybridoma that binds to human CXCR5 and cynomolgus cynomolgus monkey CXCR5

Female BALB / c mice were vaccinated three times at a three-week interval with a mixture containing 1 × 10 ⁶ BaF3 cells overexpressing human CXCR5 (SEQ ID NO: 32) or expressing human CXCR5 (SEQ ID NO: 32) Of 300.19 cells and 5 µg CPG (ODN1826) (Invivogen) adjuvant. In the final booster immunization, the MembranePro ™ functional protein expression system (Thermo Fisher) was used to immunize the 20 µg virus-like particles (VLP) prepared from HEK-293 cells overexpressing human CXCR5 (SEQ ID NO: 32) mouse. Five rounds of fusion were performed and eighty-eight pure lines that combined with human CXCR5 expressing HEK-293 cells (hCXCR5-293) were obtained. Their monoclonal antibodies were purified by protein A and tested for their binding to hCXCR5 HEK-293 and HEK-293 cells expressing cynomolgus monkey CXCR5 (SEQ ID NO: 33) (ie cynoCXCR5-293). Before staining with anti-mouse IgG PE (Southern Biotech), hCXCR5-293 or cynoCXCR5-293 cells were stained with monoclonal antibodies at 10 µg / ml. The cells were analyzed by flow cytometry using a FACSVerse analyzer (BD Biosciences). Screening for binding to hCXCR5 HEK-293 and cynoCXCR5 HEK-293 cells identified 34 antibodies that bind to both human and cynomolgus monkey CXCR5 (Figure 3).
Example 2: Anti-CXCR5 antibody binding to Raji's cells and antagonist activity in calcium flux analysis

The antibody binding to both hCXCR5-293 and cynoCXCR5-293 was further analyzed by flow cytometry using a FACSVerse analyzer (BD Biosciences, Franklin Lakes, NJ) for the binding of cells to Raji cells. The apparent affinity of the antibody to Raji's cells can be calculated as the EC ₅₀ of the equilibrium binding titration curve, where the geometric mean fluorescence intensity (gMFI) of the antigen-binding population is quantified by flow cytometry and the results are shown in Table 2 .

The data indicates that the binding of some antibodies to Raji's cells is greater / less than that of other antibodies. These data confirm that these antibodies recognize endogenous CXCR5 on Raji cells from Burkitt's lymphoma patients, indicating that the antibodies can bind B cells in vivo. In addition, the affinity of antibody 11G2 for CXCR5 on Raji cells is higher than the reference anti-CXCR5 antibody 16D7 (WO 2009/032661), indicating that 11G2 can be more potent than 16D7.

The antagonistic activity of antibodies that bind both hCXCR5-293 and cynoCXCR5-293 was also tested in calcium flux analysis. In addition, the reference anti-CXCR5 antibody 16D7 (WO 2009/032661) was included as a control. Briefly, hCXCR5 HEK-293 cells were seeded in DMEM high glucose medium in 96-well plates. After overnight incubation, the medium was removed and 100 µl of 1X Fluo-4 NW (Thermo Fisher Scientific, Waltham, MA) was added to the cells. The cells were then incubated at 37 ° C for one hour. Serial dilutions of monoclonal antibodies were added to the cells and incubated for one hour at room temperature. The calcium flux was measured by adding 111 nM recombinant hCXCL13 (BPS Bioscience, San Diego, CA) on a Flexstation (Molecular Devices, Sunnyvale, CA) to stimulate 485 nM and emit 525 nM. Collect data at 1.52 second intervals. The instrument determines the calcium flux by subtracting the lowest reading from the highest reading. The IC50 value of the ligand-induced calcium flux is based on GraphPad Prism® (version 6.0, GraphPad Software, Inc, San Diego, CA) nonlinear regression curve fitting and sigmoid log determination using an agonist dose-response model (Table 2). Inhibition of calcium flux demonstrates functional antagonism. Therefore, the 11G2 antibody is not only a potent depleting agent via ADCC, but also an antagonist that provides a second mechanism of action. The antagonism of antibody 11G2 is comparable to that of comparator antibodies such as 2C9 (reference antibody, see eg WO 2012/010582) and 16D7 (reference antibody, see eg WO 2009/032661). Analysis of cAMP reports also confirmed functional antagonism (see below).
Table 2
Example 3: Selection of anti-CXCR5 antibody heavy and light chain variable regions

The antibody-producing hybridomas analyzed in Example 2 were solubilized using RNeasy microkits (Qiagen, Hilden, Germany) and then the first strand of the cDNA used the superscript III first strand synthesis system (Thermo Fisher Scientific, Waltham, MA) synthesis. The light chain and heavy chain variable regions (VL and VH) were amplified by PCR using mouse light and heavy chain degenerate primers containing the nucleic acid sequences SEQ ID NO: 64-88 shown in Table 16. After PCR, VL and VH were cloned in Zero blunt TA vector (Thermo Fisher Scientific, Waltham, MA) and then sequenced. The amino acid sequences of mouse VL and VH of 11G2, 5H7 and 41A10 antibodies are shown in Table 16 under SEQ ID NO: 35-40. CDR is underlined.
Example 4: Defucosylated and fucosylated chimeric antibodies

To assess the effect of defucosylation and fucosylation on antibody activity, antibodies from the fucosylated and defucosylated versions of Example 3 were prepared in mammalian cells.

The VL and VH of related antibodies were further cloned into vectors containing human κ constant region and human IgG1 constant region, respectively. The variable heavy region was cloned into a pSMED2 mammalian expression vector containing a human IgG1 constant region (SEQ ID NO: 89), which produced a chimeric mouse-human full-length heavy chain. The variable light chain region was cloned into a pSMEN3 mammalian expression vector containing human kappa constant region (CK) (SEQ ID NO: 90) to prepare a chimeric mouse-human full-length light chain.

The vector containing the chimeric antibody gene was briefly transfected into HEK-293F cells (Thermo Fisher Scientific, Waltham, MA) to prepare a fucosylated chimeric antibody. The fucosylated chimeric antibody was subsequently purified using a protein A column. The apparent affinity of the chimeric antibody to Raji's cells (cell-bound EC50) was determined by flow cytometry using a FACSVerse analyzer (BD Bioscience, Franklin Lakes, NJ) and is shown in Table 3.
table 3

The defucosylated chimeric resistance system was produced using the Potelligent® CHOK1SV cell line (BioWa / Lonza, Allendale, NJ), which lacks the gene responsible for fucose addition (α-1,6-fucosyl transfer Enzyme, FUT8 ) two dual genes. The chimeric light chain containing the human kappa constant region was cloned in the Lonza pEE12.4 GS vector (Lonza Biologics, Basel, Switzerland) and the chimeric heavy chain containing the human IgG1 constant region was cloned in the Lonza pEE6.4 GS vector ( Lonza Biologics, Basel, Switzerland). The heavy chain expression cassette from pEE6.4 was purified by digesting NotI and PvuI and then cloned in the NotI and PvuI sites in pEE12.4 containing the chimeric light chain. The final vector DGV containing heavy and light chain expression cassettes was linearized with PvuI and then electroporated into Potelligent® CHOK1SV cells. After 24 hours of transfection, Potelligent® cells were seeded in CDCHO medium (Thermo Fisher Scientific, Waltham, MA) in 96-well plates at 3-5K cells / well, supplemented with 1XHT (Thermo Fisher Scientific, Waltham, MA), 1 mM uridine (Sigma, St. Louis, MO) and 50 µM MSX (EMD Millipore, Billerica, MA). After three to four weeks of incubation, antibody titers from pure line supernatants were analyzed on Octet (Pall Fortebio, Fremont, CA). Further scale up high-performing pure lines for antibody production. The defucosylated chimeric antibody was then purified on a protein A column.

To determine the specificity of the antibody for CXCR5, the binding of the antibody to cells expressing other cytokines was evaluated. In short, use 5 µg / ml or 10 µg / ml chimeric antibodies against CXCR5 chimeric antibodies on FlowCellect chemoattractant receptor cell lines (EMD Millipore, Billerica, MA) expressing CXCR1, CXCR2 or CXCR3 Carter (ATCC, Manassas, VA) stained. The cells were then incubated with goat anti-human PE-bound (Southern Biotech, Birmingham, AL), and then analyzed by flow cytometry using a FACSVerse analyzer (BD Biosciences, Franklin Lakes, NJ). As shown in FIGS. 4A-4D, all chimeric antibodies displayed and exhibited CXCR1 (FIG. 4A), CXCR2 (FIG. 4B), CXCR3 (FIG. 4C), or CXCR4 / Jurkat cells (FIG. 4D) compared to the positive control. The combination of the cells is extremely low.
Example 5: Humanization of mouse monoclonal antibodies

To avoid any possible HAMA (human anti-mouse antibody) immunogenicity, by using the human germline framework sequence from IGKV1-39 (DPK9 light chain variable domain, gene bank deposit number X93627.1, SEQ ID NO: 93) and the human germline framework sequence from IGHV3 (DP54 heavy chain variable, gene bank deposit number AB019440, SEQ ID NO: 91) humanized the 41A10 (C-41A10) and 11G2 (C-11G2) chimeric antibodies.

The humanized versions of the chimeric antibody 11G2 (C-11G2) and the chimeric 41A10 (C-41A10) are produced by complementarity determining region (CDR) grafting (hereinafter referred to as "CDR grafting"). That is, the JH4 fragment (SEQ ID NO: 92) was used to graft the heavy chain CDR to the human DP-54 framework region (VH3 subunit; SEQ ID NO: 91), and the JK4 fragment (SEQ ID NO: 94) was used The light chain CDR was grafted onto the human DPK9 framework (VKI subunit; SEQ ID NO: 93).

Link the humanized VH region to the human IgG1 constant region (SEQ ID NO: 89) and then sub-colonize in a proprietary expression vector to generate CDR grafted heavy chains, including but not limited to SEQ ID NO: 96 (11G2 CDR Graft VH). The humanized VL region is fused with the human kappa constant region (SEQ ID NO: 90) and then sub-colonized in a proprietary expression vector to produce a CDR grafted light chain, including but not limited to SEQ ID NO: 97 (11G2 CDR Graft VL).
Example 6 : Binding affinity of defucosylated or fucosylated anti- CXCR5 antibody

The apparent affinity of chimeric and humanized CXCR5 fucosylated and defucosylated antibodies to cell surface CXCR5 was evaluated. More specifically, in order to determine the apparent affinity of chimeric and humanized CXCR5 antibodies and CXCR5 expressing cells, cell binding experiments were performed on human and cynomolgus monkey peripheral blood mononuclear cells (PBMC) and human tonsil mononuclear cells (TMC) . The apparent affinity of CXCR5 mAb and CXCR5 + cells (B cells, bona fide Tfh cells and circulating Tfh-like cells) is calculated as the EC50 of the equilibrium binding titration curve, where the geometric mean fluorescence intensity of the antigen-binding population (gMFI) is Quantify by flow cytometry.

Trima® residues from healthy human donors, from Trima® dissociation collection and enrichment of PBMC were obtained from Blood Centers of the Pacific (San Francisco, CA). PBMC was separated by density gradient centrifugation using SepMate tube and Lymphoprep ^™ according to the manufacturer's instructions (STEMCELL Technologies, Vancouver, BC, Canada).

Tonsils monocytes (TMC) were isolated from human tonsils obtained from BioOptions (Brea, CA). Briefly, the tonsils are cut into small pieces (3-4 mm) using a sterile spatula in cold RPMI 1640 medium. The tonsil tissue was then digested in digestion medium (3 mL 10x collagenase, 300 mL 100x deoxyribonuclease (DNase), 6.7 mL RPMI 1640) at 37 ° C for 30 minutes. RPMI 1640 supplemented with 10% fetal bovine serum (FBS) was added to neutralize the enzyme activity and then the tissue was filtered sequentially through the nylon filter element filter (70 mm and 40 mm mesh size nylon). After centrifugation, the pellet was subjected to erythrocyte lysis using ammonium chloride dissolution buffer. Remove the slices in phosphate-buffered saline (PBS) / 2% FBS / 2 mM ethylenediaminetetraacetic acid (EDTA) using low speed centrifugation (200 × g) for 10 minutes. Using EasySep ^™ human CD4 + T cell enrichment kits, CD4 + T cells were purified into TMC mixtures according to the manufacturer's instructions (STEMCELL Technologies, Vancouver, BC, Canada).

After separation, in a 96-well U-shaped bottom culture plate, PBMC was seeded at 1.0 × 10 ⁵ cells / well in separation buffer (PBS containing 1% FBS and 1 mM EDTA) and at 2.0 × 10 ⁵ cells / well The density of wells was inoculated with CD4 + TMC. The plate was then centrifuged at 1500 rpm for 5 minutes at room temperature. PBMC and CD4 + TMC were resuspended in fluorescent activated cell sorting (FACS) buffer (PBS containing 1% FBS) and incubated on ice for 2 hours. L / well; Biolegend) and a 4-fold serial dilution of antibody (11-point dilution series, starting at 5000 ng / mL for PBMC and 312.5 ng / mL for CD4 + TMC). Subsequently, PBMC and CD4 + TMC were washed 3 times with FAC buffer and resuspended in 50-100 mL of FACS buffer, which contains fluorescent binding antibody for staining lymphocyte subpopulations and for detecting CXCR5 Fluorescent antibody (mAb) binds secondary antibody to human Ig. After incubation at 4 ° C for 30 minutes, PBMC and CD4 + TMC were washed twice with FACS buffer and resuspended in 0.5% paraformaldehyde (PFA) in 125 mL PBS. The culture plates were stored at 4 ° C until analyzed by flow cytometry (BD LSRFortessa ^™ Cell Analyzer, BD Biosciences, Franklin Lakes, NJ).

Cell binding titration curves were generated by gMFI plotting the logarithm of the antigen binding population to the CXCR5 antibody concentration. The EC ₅₀ value was determined using GraphPad Prism ^® (Version 6.0, GraphPad Software, Inc, San Diego, CA) nonlinear regression curve fitting and the sigmoid logarithm of the agonist dose-response model:
Log ( agonist ) versus response - different slopes ( four parameters )
Y = Bottom + (Top-Bottom / (1 + 10 ^ ((LogEC ₅₀ -X) * Hill slope )))

Where Y is gMFI, X is antibody concentration, Top is the maximum Y value in the upper plateau of the S-shaped curve, Bottom is the minimum Y value in the plateau of the lower S-shaped curve, and LogEC ₅₀ is the inverse of the curve Logarithm of antibody concentration at the curve point.

Using the average value and standard deviation (STDEV) is repeated a plurality of times through ₅₀ summarize the experimental values EC, which the. The results (ie, the average apparent affinity of fucosylated or defucosylated chimeric and humanized CXCR5 mAbs to CXCR5 expressing cells) are shown in Table 4. The results also include affinity binding data obtained using anti-CXCR5 control antibodies 2C9 (WO 2012/010582), 16D7 (WO 2009/032661) and 11A7 (WO 2016/028573) (including comparisons). It should be noted that there are multiple VL and Fucosylated and defucosylated chimeric and humanized 11G2 antibodies for VH combinations (i.e. h11G2 XC51 / XC152, h11G2 XC153 / XC155, h11Gh11G2 XC153 / XC156, h11G2 XC154 / XC155 and h11G2 XC154 / XC157) Has an apparent affinity for human B cells that is approximately equal. That is, the data show that defucosylation does not seem to affect the affinity of the antibody for CXCR5 expressing cells. In addition, the data confirmed that the h11G2 XC154 / XC155 antibodies (fucosylated and defucosylated) exhibited an affinity of about 10 times higher than that of the control anti-CXCR5 antibody 2C9 and about 100 times higher than that of 11A7. The control anti-CXC5 antibody 16D7 did not display saturable binding. These data indicate that defucosylation does not affect the affinity of 11G2 antibody for binding to CXCR5 expressing cells. Because affinity is a measure of the strength of the interaction between the epitope and the antigen binding site (ie, paratope) of the antibody, these data indicate that even though the antibody binds a similar epitope, it does not Strength binding epitope.
Table 4
Example 7 : Concentration-dependent binding of defucosylated or fucosylated humanized 11G2 antibody compared to fucosylated reference antibody

Humanized fucosylation 11G2 (h11G2 VL XC154 / VH XC155 also known as h11G2 154/155 or h11G2 XC154 / XC155) and fucosyl humanized 11G2 CXCR5 (fucosyl h11G2 154/155) and from The concentration-dependent binding of B cells of human PBMC was compared with the binding of the comparison agents mAb 2C9, 16D7 and 11A7. The cell binding titration curve was generated by plotting the log-binding gMFI of the antigen-binding population against the CXCR5 antibody concentration and is shown in FIG. 5.

The data indicate that the fucosylated and defucosylated h11G2 154/155 have the same curve, indicating that there is or is present on the N-linked glycoform present at Asn297 on the constant chain of at least one but preferably these two antibodies or The absence of fucose does not affect the affinity of the antibody for CXCR5 present on the cell.

Further, the data further indicate that fucose glycosylated (filled circles, 9.630 × 10 ^{- 12} M) and de-glycosylated fucose (open ^{circles, 1.188 × 10 - 11 M)} h11G2 154/155 EC50 antibody far lower than the comparison EC50 antibody agents: 2C9 (solid ^{triangles, 6.24 × 10 - 11),} 11A7 ( filled diamonds, 9.265 × ^{10 -} 10) and 16D7 (solid squares, approximately 0.004945 and which is not saturated). These data confirm that the h11G2 154/155 antibody has different binding characteristics than the comparative antibody. These data therefore indicate that the 11G2 antibody and 2C9, 11A7 and 16D7 do not bind the same epitope on CXCR5.
Example 8: ADCC activity of chimeric and humanized CXCR5 antibodies

To determine the efficacy and efficacy of a group of chimeric and humanized CXCR5 antibodies to stimulate activity-dependent cellular cytotoxicity (ADCC), serial dilutions of CXCR5 antibodies or isotype controls were compared with those from healthy human donors or cynomolgus monkeys Peripheral blood mononuclear cells (PBMC) are incubated together. In this analysis, the PBMCs are the source of natural killer (NK) effector cells and target B cells and Tfh-like cells. Flow cytometry was used to quantify the number of B cells and Tfh-like cells remaining after approximately 20 hr. Similarly, the ability of humanized antibodies to induce ADCC from bona fide Tfh cells from human tonsils was evaluated using CD4 + T cells isolated from tonsil monocytes and addition of NK cells isolated from PBMC.

As described in Example 6, Trima® residues from healthy human donors, from Trima® dissociation collection, and enrichment of PBMC were obtained from the Pacific Blood Center (San Francisco, CA). PBMC was separated by density gradient centrifugation using SepMate ^™ tube and Lymphoprep ^™ according to the manufacturer's instructions (STEMCELL Technologies, Vancouver, BC, Canada). After separation, PBMC were inoculated in a complete RPMI medium in a U-bottom 96-well plate at a density of 2.0 × 10 ⁵ cells / well.

In this analysis, the PBMCs are the source of natural killer (NK) effector cells and target B cells and Tfh-like cells. A serial dilution of CXCR5 mAb or isotype control was added to the wells and incubated at 37 ° C, 5% CO2 for approximately 20 hours. The plate was then centrifuged at 1800 rpm for 5 minutes at room temperature (RT). The PBMCs were then washed with ice-cold FACS and resuspended in 50 µl of ice-cold FAC buffer containing fluorescent-bound antibodies for staining lymphocyte subpopulations. After incubation at 4 ° C for 15-30 minutes, PBMC was washed twice with ice-cold FACS buffer and resuspended in 0.5% paraformaldehyde (PFA) in 100 µL PBS. CountBright ^™ absolute counting beads (Thermo Fisher Scientific) were added to each well (15 microliters / well). The culture plates were stored at 4 ° C until analyzed by flow cytometry (BD LSRFortessa ^™ cell analyzer).

Tonsillary monocytes (TMC) were also isolated as described in Example 6 above.

The cytotoxicity titration curve was prepared according to the method described for Example 6. Using the average value and standard deviation (STDEV) is repeated a plurality of times through ₅₀ summarize the experimental values EC, which the. The results are shown in Table 5. Includes data obtained using anti-CXCR5 reference antibodies 2C9, 16D7, and 11A7 for comparison.
table 5
Average cytotoxicity of chimeric and humanized CXCR5 mAb on CXCR5 expressing cells

The data disclosed herein shows that even the fucosylated h11G2 154/155 antibody has an ADCC activity (253.72 ± 672.14) greater than that of the comparator antibodies 2C9 (380.31 ± 757.04) and 16D7 (2590.61 ± 6343.88). Fucosylated antibody 11A7 exhibits greater ADCC activity than any fucosylated antibody tested. However, the data shows that defucosylated h11G2 154/155 has higher ADCC activity than any antibody tested (including fucosylated 11A7). The characterization studies presented here show that chimeric CXCR5 mAbs 41A10 and 11G2 trigger ADCC of human B cells with EC50s of 11.73 nM and 0.56 nM, respectively. The fucosyl version of the chimeric CXCR5 mAbs 41A10 and 11G2 increased the potency of ADCC activity by at least about 100-fold. Humanized variants trigger ADCC of human B cells with an efficiency comparable to or better than that of their corresponding chimeric counterparts. Like the chimeric CXCR5 mAb, fucosylated humanized variants increase the effectiveness of ADCC by at least about 100-fold compared to its general fucosylated counterpart. Fucosyl h11G2 is more potent in terms of ADCC than the fucosylated versions of 2C9 and 16D7, and fucosyl h11G2 is at least equivalent to fucosylated 11A7.
Example 9: 11G2 antibody antagonizes CXCL13 signaling

To determine whether the CXCR5 anti-system functionally antagonizes CXCL13 signaling, cyclic adenosine monophosphate (cAMP) report analysis was used in engineered cell lines that stably express human CXCR5. In these cells, CXCL13 inhibits forskolin-triggered cAMP production in a concentration-dependent manner.

To determine whether the CXCR5 system antagonized CXCL13 signaling, Hit Hunter® cAMP analysis (DiscoveRx Corporation, Fremont, CA) was performed in CHO-K1 cells that stably express human CXCR5. In this analysis, active b-galactose (b-gal) is formed by the complementation of enzyme fragments during the production of cAMP. The b-gal can then convert the chemiluminescent matrix to produce an output signal that can be detected on a standard microplate reader. CXCR5 ligand CXCL13 inhibits forskolin (20 μM) -induced cAMP production in a concentration-dependent manner. To test the efficacy and efficacy of CXCR5 antibodies in inhibiting CXCL13-mediated forskolin-induced cAMP production, each CXCR5 antibody was tested in the presence of forskolin (20 μM) and CXCL13 (with its IC _{80 of} 600 pM). Serial dilutions were added to CHO-K1 cells expressing CXCR5.

A functional antagonism titration curve was prepared as described for Example 6 (including EC ₅₀ value determination). The results are shown in Table 6.
Table 6 In vitro stimulation of CXCL13 inhibited cAMP production in CXCR5 + CHO-K1 cells

In an independent study, the functional antagonism of fucosylated humanized 11G2 154/155 was tested and its performance was similar to that of humanized fucosylated 11G2 154/155 (ie EC ₅₀ = 961.4 pM).

The data shows that h11G2 154/155 inhibits CXCL13 signaling at least as much as the reference antibodies 2C9, 16D7, and 11A7. In fact, h11G2 inhibited CXCL13 signaling (120.6%) significantly more than 16D7 (102.4%) and 11A7 (105.9%). These data indicate that the 11G2 antibody can be a useful novel therapeutic agent for the treatment of diseases, disorders or conditions mediated by or related to CXCL13 signaling mediated by CXCR5.
Example 10: Locating the binding site on hCXCR5 of 11G2 antibody

The fucosyl humanized 11G2 154/155 binding site on CXCR5 was identified by grafting the extracellular N-terminal amino acid from mouse CXCR5 onto human CXCR5.

More specifically, the localization strategy utilizes the fact that 11G2 binds to human CXCR5 (SEQ ID NO: 32), but not mouse CXCR5 (SEQ ID NO: 34). The alignment of the amino acid sequences of human CXCR5 (hCXCR5) and mouse CXCR5 (mCXCR5) is shown in FIG. 6. Amino acid residues in the extracellular domain of CXCR5 are indicated by underlining. The extracellular domains (ECD) of mouse and human CXCR5 proteins are indicated in bold under the sequence of each region in FIG. 6 and labeled as "N", "L1", "L2", and "L3". These ECDs were swapped between mouse and human proteins to identify the regions responsible for the specificity of 11G2 for human CXCR5 and the lack of binding to mCXCR5. Each of the other regions from mouse CXCR5 is exchanged with the same region in human CXCR5 to prepare chimeric proteins called XC251, XC252, XC253, XC254, XC255 and XC256, where the human CXCR5 protein contains a specific counterpart Mouse CXCR5 domain. Table 7 provides a search table showing the mouse (m) region exchanged for human CXCR5.
Table 7
(Mouse / human chimeric CXCR5 protein with ECD exchanged)

More specifically, the mouse L3 domain was replaced with the corresponding human L3 domain to prepare the chimeric protein XC251, which contains the mouse L3 domain in the case of the human CXCR5 protein (hCXCR5-mL3). Similarly, the mouse L2 was replaced with the corresponding human domain to prepare the chimeric protein XC252 (hCXCR5-mL2); the mouse N domain was replaced with the corresponding human N domain to prepare the chimeric protein XC253 (hCXCR5-mN ); Replace the mouse L2 and L3 domains with corresponding human domains to prepare the chimeric protein XC254 (hCXCR5-mL2-mL3); replace the mouse mN and L3 domains with corresponding human domains to prepare the chimeric Protein XC255 (hCXCR5-mN-mL3); and the mouse mN and L2 domains were replaced with corresponding human domains to prepare the chimeric protein XC255 (hCXCR5-mN-mL2).

Evaluate the binding of 11G2 to mouse CXCR5, human CXCR5, and various ECD-switched mouse-human chimeric CXCR5 proteins (XC251-XC256) and compare their binding to various antibodies, which are also rat anti-mouse CXCR5 (Rat) (Cat. No. MAB6198, R & D System, Minneapolis, MN), Rabbit anti-human CXCR5 (Rb) (Cat. No. ab46218, Abcam, Cambridge, MA), 2C9, 16D7. The results are shown in Table 8. Bold numbers indicate antibodies that bind to proteins.
Table 8
(FACS analysis of the binding of antibody to ECD-transforming mouse-human CXCR5 chimeric protein)

The data shows that in the case where the antibody is specific for mouse CXCR5, as long as the chimera contains the mouse N domain, it binds to the mouse-human chimeric CXCR5 that transposes ECD. Similarly, for antibodies that bind to human CXCR5 (11G2, 2C9, 16D7, and Rb), if the human N domain is present in the chimera, the antibody binds to chimeric CXCR5. Therefore, the species specificity of antibodies appears to be driven by binding localized in the N domain. More importantly, these data show that the N domain of human CXCR5 is required for the binding of antibodies 11G2, 2C9 and 16D7.
Example 11: Identification of amino acid residues unique to the binding of 11G2 antibody to hCXCR5

To more accurately determine the specific amino acid residues that are critical for the binding of the antibody to hCXCR5 and to further distinguish anti-human CXCR5 antibodies, a more detailed study of the N domain of CXCR5 was conducted. More specifically, point mutations were made in the N-domain fragment of human CXCR5 (amino acids 1-58 numbered according to SEQ ID NO: 32) to prepare the corresponding mouse amino acid residues each containing CXCR5 Point mutant protein. Stable transfectants expressing each mutant protein were generated and the cells were tested for antibody binding using flow cytometry. That is, nineteen (19) human CXCR5 N domain mutant proteins (referred to as XC276-XC294) each containing a single amino acid substitution were prepared, in which the human residues of CXCR5 were replaced with corresponding mouse residues, as shown in the table Explained in 9. The human CXCR5 mutein further contains the FLAG epitope (DYKDDDDK) shown in lower case in Table 9 at the N-terminus to allow regularization based on performance levels; FLAG markers do not affect antibody binding to the mutein. Generally, humans do not substitute positions where mouse amino acid residue substitutions are conservative substitutions, and these residues are indicated in italics.

Table 9 shows the amino acid sequence of the N region of hCXCR5 compared to the amino acid sequence of the N region of mouse CXCR5 (showing the first 58 amino acids). The amino acids that differ between the two sequences are underlined. The diagram also shows the various constructs prepared (XC276-XC294), in which mouse amino acid residues are replaced with corresponding human amino acids in hCXC5. For example, XC276 is a human CXCR5 with a single amino acid substitution that changes D (human) to G (mouse). Because no change occurred, the rest of the hCXCR5 amino acid sequence (SEQ ID NO: 32) was not displayed. Because the antibody tested did not bind to mouse CXCR5, muteins were prepared to determine the amino acid residues that are critical for antibody binding to hCXCR5.

The nucleic acid encoding each mutant peptide was transiently transfected into HEK-293T cells. 2 days after transfection, collect the cells and use 1 µg / ml anti-FLAG PE (Biolegend) or 1 µg / ml or 3 µg / ml fucosyl humanized 11G2 154/155, 2C9, S16D7 or 11A7, and then anti-human IgG PE (Southern Biotech, Birmingham, AL) staining. The cells were analyzed by flow cytometry using Accuri (BD Biosciences Franklin Lakes, NJ). The geometric mean fluorescence intensity (gMFI) was measured and the ratio of each CXCR5 antibody to anti-flag gMFI was calculated.

As shown in Figure 7, all four antibodies bind to wild-type human CXCR5 (XC275; WT). Antibodies then provide different modes of binding various point mutant peptides. That is, 2C9 does not bind XC276 (including the mutation D10G0) or XC277 (including the insertion of amino acid residues S and I into the human CXCR5 N domain), while 11G2, 16D7, and 11A7 bind their proteins. These data indicate that inserting these two amino acids into the hCXCR5 sequence eliminates 2C9 binding but does not affect the binding of three other antibodies, indicating that the epitopes of 2C9 are different from those of 11G2, 16D7, and 11A7. More importantly, mutation of leucine (L) to threonine (T) at amino acid residue position 11 (based on the numbering of the sequence SEQ ID NO: 32) only completely eliminated the binding of 11G2 to XC278 but did not affect the antibody 2C9, 16D7 or 11A7 bind to this protein. Therefore, the leucine residues present at position number 11 (relative to the amino acid sequence set forth in SEQ ID NO: 32) appear to be essential for the binding of 11G2 to human CXCR5, but for 2C9, 16D7 or The combination of 11A7 and hCXCR5 is not important. These data show that the epitopes of 11G2 are different from those of their antibodies. In addition, Figure 7 shows that all four antibodies bind XC279, such that at amino acid residue number 12 (relative to the sequence SEQ ID NO: 32) E is replaced by Y. For any of these antibodies to bind to hCXCR5 It's not critical.

The binding of the four antibodies to XC280 (which contains the substitution W for the amino acid (K) at amino acid residue number 19 (according to the sequence SEQ ID NO: 32)) results in the binding of XC280 to antibodies 16D7 and 11A7 Loss, but does not affect the binding of antibodies 11G2 and 2C9 to the mutant protein (Figure 7). These results further confirm that these four antibodies do not share the same epitope on hCXCR5.

Figure 7 also shows that changing the D at position 22 (according to the numbering of SEQ ID NO: 32) to alanine (A) completely abolishes the binding of 11G2 to the XC281 mutant protein, but does not affect the binding of antibodies 2C9, 16D7 and 11A7. This further emphasizes that the epitope of 11G2 is different from that of antibodies 2C9, 16D7 and 11A7.

All four antibodies bind equally to XC285-294 and their binding is not affected by any of the amino acid substitutions in these mutant proteins. These data indicate that for antibody 11G2, 2C9, 11A7 and 16D7, the amino acid residues at these positions (27, 28, 30, 31, 33, 35, 36, 37, 39 and 40; see Table 9) Did not participate in antibody binding to CXCR5.

Therefore, these data indicate that the leucine (L) residue at amino acid position 11 and the aspartic acid (D) residue at amino acid position 22 (both relative to the amino acid sequence (Number of SEQ ID NO: 32) is essential for the binding of the 11G2 antibody to hCXCR5, because the corresponding mouse amino acid residues are substituted for any of these residues (or both) at their positions Eliminate the binding of antibody to hCXCR5. These data also show that the epitope of antibody 11G2 is different from that of antibody 2C9, 16D7 or 11A7, because the change of L11 and / or W22 does not affect the binding of these antibodies to hCXCR5.

Figure 7 also shows that all four antibodies bind the mutant CXCR5 proteins XC282 to XC284. All four antibodies also bind proteins XC285 to XC294. These data indicate that the epitopes of antibodies 11G2, 2C9, 16D7 and 11A7 are not in the region of the N domain of hCXCR5 indicated by the mutant proteins XC282 to XC294.
Example 12 : 11G2 is selective for CXCR5 and does not bind other members of the GPCR family of chemokines

Beta-inhibitory protein coupling analysis (DiscoveRx) was used to assess the selectivity of fucosyl humanized 11G2 154/155 to twenty (20) members of the chemoattractant GPCR family. This analysis uses whole cells stably transfected with each receptor, and directly measures GPCR activity by detecting the interaction of β-inhibitors with activated GPCRs. Because inhibitor protein recruitment is not related to G protein signaling, these analyses provide a universal screening and profiling platform that can be used for virtually any Gi, Gs, or Gq-coupled receptor, providing a Standardized and effective comparison (regardless of G protein coupling). The analysis was performed in agonist (no ligand present) and antagonist mode (in the presence of ligand under EC90). Include the following chemokine receptors in the selective panel: CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CMKLR1, CXCR3R1, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, CXCR7, XCR1.

When tested in agonist mode, no significant activity of fucosyl humanized 11G2 154/155 (tested at 200 nM) on any other chemokine receptor was observed and compared to the antagonist mode. Activity of all other chemoattractant receptors, which has significant activity only as an antagonist of CXCR5 (92% inhibition of ligand-induced β-inhibitory protein coupling), ie, for any other chemoattractant tested No significant inhibition was observed in the hormone receptor.
Example 13 : h11G2 does not show multi-reactivity to DNA , insulin or LPS

Use multi-reactivity analysis to determine whether the anti-system may react with multiple unrelated antigens in vivo. The lack of polyreactivity indicates that 11G2 is a CXCR5-specific antibody and it does not bind to non-target targets in vivo.

Black DELFIA culture plates (Thermoscientific) coated with 10 µg / ml double stranded DNA (dsDNA; Millipore), 5 µg / ml lipopolysaccharide (LPS; Sigma), 10 µg / ml insulin (Sigma, St. Louis, MO ) Or phosphate-buffered saline (PBS) and incubated overnight at 4 ° C. The culture plate was then washed with a Biotek ELx405 microplate washer (Biotek, Winooski, VT) with water and then capped with 200 µl of analysis buffer containing 1 × PBS, 0.05% Tween 20 and 1 mM EDTA at room temperature for one hour. After an additional washing step, the culture plate was incubated with a serial dilution of anti-CXCR5 antibody (all antibodies were fucosylated) at room temperature for one hour. For detection, 100 µl of DELFIA-Eu-N1-anti-human IgG (50 µg / ml; Perkin Elmer, Waltham, MA) was added to the culture plate and incubated for one hour at room temperature. Finally, 100 µl of DELFIA enhancement solution was added to each well and the culture plate was shaken at room temperature for 15 minutes before reading on a VictorX4 multi-label plate reader (PerkinElmer, Waltham, MA) under europium fluorescence. The information is shown in Table 10.
Table 10
Fluorescence for CXCR5 mAb multi-reactivity (counting)

In an independent study, tested the fucosylation of defucosylated humanized 11G2 154/155 and did not show binding to insulin, DNA, LPS, or PBS, and the fucosyl group shown in Table 10 above The results of the humanized 11G2 154/155 are consistent.

These data show that 11G2 does not react with insulin, DNA or LPS, and is less reactive than the comparator antibodies 2C9, 16D7 and 11A7. These data indicate that the 11G2 series may be useful novel therapeutic agents. Polyreactive antibodies bind a series of biomolecules in a non-specific manner by definition. The absence of 11G2 in the general multi-reactivity analysis further indicated that 11G2 is specific for CXCR5. PK, TK and toxicity data also show that 11G2 is specific for CXCR5.
Example 14: Overview of differences between 11G2 and comparator antibodies

Table 11 below summarizes the characteristics of 11G2 and various comparator antibodies previously described elsewhere herein. ND means not determined.

These data indicate that the fucosylated and defucosylated antibodies 11G2 are significantly different from the comparator antibodies 2C9, 16D7 and 11A7. That is, 11G2 binding is eliminated by the substitution of mouse amino acid residues at positions 11 and 22, while the binding of the comparator antibody is not affected by the substitution of these residues (numbered according to the amino acid sequence SEQ ID NO: 32) . In addition, the binding of 2C9 requires the presence of amino acid residues W19, D22, and R23 to bind to hCXCR5, but none of these antibodies requires all three of these amino acid residues, even though 11A7 binds to hCXCR5 through position 19. (According to the amino acid sequence SEQ ID NO: 32 number) alanine substitution to W elimination. Unlike all other antibodies, the insertion of two mouse residues between D10 and L11 of human CXCR5 (based on the amino acid sequence SEQ ID NO: 32) eliminates the binding of 16D7 to hCXCR5 (see Figure 7). Therefore, the data provided herein indicate that 11G2 has different epitopes than antibodies 2C9, 16D7, and 11A7, and that these antibodies have different epitopes.

In addition, the information provided in this article shows that 11G2 has an affinity for hCXCR5 (expressed as EC50) (7.08 pM) that is at least 10 times greater than 2C9 (65.59 pM) and at least 100 times greater than 11A7 (563.63 pM), and is 16D7. (It cannot be compared because it is not saturated.) The difference is huge.
Table 11

All these data indicate that 11G2 is substantially different from the comparative agents 2C9, 16D7 and 11A7, and is a possible novel and useful therapeutic agent for the treatment or prevention of biological activity of CXCR5 (including but not limited to CXCR5 mediated CXCL13 Signaling) a disease, disorder or condition that is mediated or associated with it. This is particularly the case for the defucosylated version of 11G2 that exhibits greatly enhanced ADCC activity, thereby enhancing its potential usefulness as a therapeutic agent in the treatment of cell expression mediated by or associated with it, such as CXCR5.
Example 15: In vivo pharmacodynamics of defucosylated h11G2 XC154 / XC155

In vivo, doses in the IV or SC range of 0.001 to 400 mg / kg / dose have been explored for fucosyl-based humanized 11G2 154/155 in multiple crab-eating macaque studies. As used in this experiment, the IV and SC formulations contained the following components: 50 mg / mL of the CXCR5 antibody or antigen-binding fragment of the invention, 20 mM histidine, 8.5% sucrose, and 0.02% polysorbate 80, 0.005 % EDTA at pH 5.8. Table 12 below describes the in vivo study design for the study of defucosylated 11G2 XC154 / XC155 in cynomolgus macaques.
Table 12

Defucosylated h11G2 VL XC154 / VH XC155 (fucosyl h11G2 XC154 / XC155) single doses in the range of 0.001 to 0.2 mg / kg B cells and peripheral blood in cynomolgus monkeys were observed Dose-dependent depletion of Tfh-like cells; the lowest dose tested (0.001 mg / kg) resulted in approximately 50% depletion of B cells and Tfh-like (ie cTfh) cells in peripheral blood.

In exploratory and key GLP toxicity studies in cynomolgus macaques, the fucosyl h11G2 XC154 / XC155 dose of ³5 mg / kg IV resulted in significant depletion of B cells and Tfh-like cells in peripheral blood as well as spleen and other lymphoid tissues (Axillary and mesenteric lymph nodes and intestinal-associated lymphoid tissues) The cellular content of lymphoid follicles is reduced; these effects are related to the pharmacologically-mediated reduction of cellular content in the B-cell region observed by immunohistochemistry. In exploratory and critical studies, partial to full recovery of B cells, Tfh-like cells and Tfh cells was observed. The dynamics of exhaustion and subsequent flooding are related to the exposure level of fucosyl h11G2 XC154 / XC155.

To determine whether the fucosyl h11G2 XC154 / XC155 system impairs humoral memory response, a vaccine recall response study was conducted in cynomolgus monkeys. Briefly, fucosyl h11G2 XC154 / XC155 was given by IV rapid injection at the level of 2 and 10 mg / kg / dose on Days 1 and 8 and TT (tetanus toxoid) was inoculated before the study Crab-eating macaques (N = 6 / group). The administration of fucosyl h11G2 XC154 / XC155 is well tolerated and results in the expected pharmacology (ie, a decrease in circulating B cells and Tfh-like cells). 7 days after the second TT vaccination, an increase in memory response of immunoglobulin M (IgM) and IgG was observed in all animals in all dose groups on day 22 compared to day 15. In animals dosed with fucosyl H11G2 XC154 / XC155 (10 mg / kg / dose), these reactions were reduced. Compared with 72,000 in the control group, the average peak anti-TT IgG titration concentration was 42,667, and At 3500 in the control group, the peak anti-TT IgM average titration concentration was 1583. The B-cell depleting antibody Rituxan® (rituximab) used as a comparison agent does not affect the anti-TT IgG memory response. In conclusion, these data show that the fucosyl h11G2 XC154 / XC155 impairs the humoral memory response in vivo.
Safety Pharmacology

Incorporate ECG and heart rate measurements into the design of key toxicity studies. No abnormal ECG was found, which can be attributed to the administration of fucosyl h11G2 XC154 / XC155. All ECGs were qualitatively and quantitatively within normal limits and there was no arrhythmia.
Example 16: In vivo pharmacokinetics and metabolism of defucosylated h11G2 XC154 / XC155

Validate electrochemiluminescence (ECL) analysis to detect the presence of ADA in cynomolgus monkey serum on the Meso Scale Discovery® (MSD) analysis platform. A positive control anti-CXCR5 anti-idiotype antibody was incorporated into cynomolgus monkey serum, and a negative control consisting of pooled normal cynomolgus monkey serum was included on each plate to monitor analytical performance. The biotin-labeled fucose h11G2 XC154 / XC155 and ruthenium-labeled fucose h11G2 XC154 / XC155 were incubated with the research sample, positive control and negative control. The fucosyl h11G2 XC154 / XC155 antibody present in the sample must be combined with the biotin-labeled and ruthenium-labeled form of the fucosyl h11G2 XC154 / XC155 to be detected in this analysis. The complex was captured via biotin-labeled fucosyl h11G2 XC154 / XC155 to a streptavidin-coated MSD multi-array culture plate and read on the MSD Sector Imager instrument. ECL signal (RLU) was generated in the MSD Sector Imager instrument by final detection using ruthenium-labeled fucosyl h11G2 XC154 / XC155 and tripropylamine.

A stratified strategy is used to test research samples for ADA. In the screening analysis, the sample is first tested with a dilution factor of 75. Samples that produced RLU below the analytical cut point were reported as negative (<1.88). Re-analyze samples in the complete dilution series that produce RLUs at or above the analytical cut-off point to determine antibody titers. Antibody titer is defined as the reciprocal dilution that will produce a sample equal to the cut point RLU. Report the logarithm of his valence (base 10).

Based on the comparison of the results of the sample collected before the administration on day 1 and the sample after administration, a conclusion about ADA induction was drawn. If the sample before the test is negative for ADA and the corresponding sample after the test is positive, the animal is considered to be positive for ADA induction. If the test samples are positive for ADA before and after dosing, it is considered that only animals with a potency of at least 0.48 (log 3, continuous dilution factor) after dosing or higher than the potency of the sample before dosing The induction was positive.
Single dose pharmacokinetics

After a single IV dose of fucosyl h11G2 XC154 / XC155 at 0.001, 0.002, 0.005, 0.1 or 0.2 mg / kg, characterize the fucosyl h11G2 XC154 / XC155 PK as part of the study to assess male and female diets The depletion and overflow of peripheral blood B cells and follicular T helper (Tfh) -like (Tfh-like) cells in crab macaques (n = 1 / gender / dose group). As used in this experiment, the IV and SC formulations contained the following components: 50 mg / mL of the CXCR5 antibody or antigen-binding fragment of the invention, 20 mM histidine, 8.5% sucrose, and 0.02% polysorbate 80, 0.005 % EDTA at pH 5.8.

After a single IV administration, the systemic exposure increased with increasing dose, and the fucosyl h11G2 XC154 / XC155 PK was characterized by low CL and low V _ss , the average of all doses was about 0.2 to 2.6 mL / h / kg and 0.03 to 0.1 L / kg. The average t½ value for all doses is in the range of approximately 1 to 4 days. At low doses (0.001-0.005 mg / kg), signs indicate a high CL, which may be due to antibody-dependent cytotoxicity (ADCC) -mediated cell depletion, which may in turn lead to fucose h11G2 XC154 / XC155 / CXCR5 complex removal (Kryzanski et al., 2016, J. Pharmacokinet. Pharmacodyn. 43 (5): 513-527; Wang et al., 2010, AAPS J. 12 (4): 729-740). At low doses, a similar clearance mechanism / non-linearity can be seen in humans. As previously described elsewhere in this article, dose-dependent depletion of B cells and Tfh-like cells in peripheral blood was observed, and the depletion of circulating B cells was more stable than that of Tfh-like cells.
Dynamics of Repeated Dose Toxicology in Crab-eating Macaques ( TK )

As part of GLP repeated dose toxicity studies, male and female cynomolgus cynomolgus monkeys (n = 3 or 4 / sex / dose group) were given 5 (IV), 20 (SC) or 200 (IV) mg / kg every two Weekly (every 2 weeks) IV or SC administration (a total of 5 administrations) fucosyl h11G2 XC154 / XC155 followed by TK and ADA assessment.

In the samples collected and analyzed from the vehicle control group, there was no quantifiable concentration of fucosyl h11G2 XC154 / XC155. In all dose groups, there was no significant gender-related difference in systemic exposure; therefore, the combined data of male and female cynomolgus cynomolgus monkeys were used to present the group average TK parameters (Table 13).

After administration of 5 (IV), 20 (SC), or 200 (IV) mg / kg every two weeks, systemic exposure was higher after repeated administrations, cumulative ratio (Study Day 43 / Study Day 1) In the range of about 1.4 to 1.7. In addition, systemic exposure increased in a dose proportional manner after IV administration. The last quantifiable concentration of fucosyl h11G2 XC154 / XC155 in the recovered animals from the 5 mg / kg (IV) dose group was observed from the 148th study day to the 281th study day of the 11-month recovery period. After SC administration, the bioavailability of fucosyl h11G2 XC154 / XC155 is estimated to be at least about 50%.

After administration of fucosyl h11G2 XC154 / XC155 at 5 (IV), 20 (SC) or 200 (IV) mg / kg, respectively, the incidence of ADA on fucosyl h11G2 XC154 / XC155 was 19% (3 / 16 animals), 17% (1/6 animals) and 0% (0/6 animals), and compared with ADA-negative animals, the serum concentration on study day 43 is generally lower among ADA-positive animals . It should be noted that the circulating concentration of fucosyl h11G2 XC154 / XC155 can interfere with the detection of ADA.
Table 13
distributed

In non-clinical species, no protein binding and tissue distribution studies were carried out on fucosyl h11G2 XC154 / XC155. After a single IV administration, the V _{ss of} fucosyl h11G2 XC154 / XC155 in cynomolgus monkeys is in the range of approximately 0.03 to 0.1 L / kg, consistent with the limited distribution expected for IgG (Lin et al., 1999, J. Pharmacol. Exp. Ther. 288 (1): 371-378; Mascelli et al., 2007, J. Clin. Pharmacol. 47 (5): 553-565).
metabolism

No fucosyl h11G2 XC154 / XC155 was used for metabolic studies. Similar to other therapeutic proteins with molecular weights higher than the glomerular filtration cutoff, it is expected that fucosyl h11G2 XC154 / XC155 is mainly metabolized by catabolism (Lobo et al., 2004, J. Pharm. Sci. 93 (11) : 2645-2668; Mascelli et al., 2007, see above; Vugmeyster et al., 2012, World J. Biol. Chem. 3 (4): 73-95).
Pharmacokinetic drug interaction

No pharmacokinetic drug interactions were performed in vitro or in vivo. The fucosyl h11G2 XC154 / XC155 is a humanized monoclonal antibody (mAb) against CXCR5 and has been shown to modulate cytokine release in vitro, but not in vivo. It has been confirmed that cytokines regulate the expression of cytochrome P450 (CYP) enzymes and transporters (Lee et al., 2010, Clin. Pharmacokinet. 49 (5): 295-310; Mahmood & Green, 2007, J. Clin. Pharmacol. 47 (12): 1540-1554). Therefore, treatment with fucosyl h11G2 XC154 / XC155 may affect the levels of CYP enzymes and transporters and thus regulate the clearance of co-administered drugs, which are substrates of these enzymes or transporters. However, the interleukin-mediated drug interactions observed for other drugs in the clinic have been moderate, resulting in less than 2-fold changes in the exposure of co-administered small molecule drugs (Huang et al., 2010, Clin. Pharmacol . Ther. 87 (4): 497-503; Evers et al., 2013, Drug Metab. Dispos. 41 (9): 1598-1609). Therefore, without wishing to be bound by any particular theory, if the combination of drugs changes the target performance, it may affect the PK of fucosyl h11G2 XC154 / XC155.
Human pharmacokinetic prediction

The PK profile of fucosyl h11G2 XC154 / XC155 in cynomolgus monkeys is typical for human IgG1 mAb in monkeys. Therefore, the predicted two-compartment PK parameter value in humans is expected to be the same as the two-compartment PK parameter value of the typical therapeutic agent IgG1 mAb and is considered to be linear in the dose range to be tested. The PK parameter values used are similar to those previously reported (Singh et al., 2015, Developability of Biotherapeutics: Computational Approaches, S. Kumar, edited by Singh S. Kumar, CRC Press). These parameters are as follows: central volume (V _c ) is 3.2 L; peripheral volume (V _p ) is 2.2 L; central clearance (CL _c ) is 0.25 liters / day; distributed clearance (Q) is 0.45 liters / day; SC the absorption rate constant (k _a) 0.26 1 / day and SC bioavailability of 60%. Based on the data disclosed herein, the predicted human serum half-life of defucosylated h11G2 XC154 / XC155 is about 17 days.
Human effective dose prediction

A model-based approach was adopted to characterize the relationship between dose, fucosyl h11G2 XC154 / XC155 concentration, and the regulation of B and Tfh-like cells in the serum of cynomolgus monkeys. Next, monkey and human pharmacokinetics / pharmacodynamics using the disclosed SBI-087 (humanized small module immunopharmaceutical (SMIP) that depletes B cells like rituximab binding to CD-20 on B cells) (PK / PD) Data conversion of fucosyl h11G2 XC154 / XC155 B cell depletion parameters from monkey to human (Cohen et al., 2016, Clin. Ther. 38 (6): 1417-1434; Dunussi-Joannopoulos et al. , 2008, Ann. Rheum. Dis. 64 (Suppl II): 190 (Abstr THU0171)). Unlike B cells, no human data is available for Tfh-like depletion; therefore, it is assumed that the translation of Tfh-like cell depletion is similar to B cells. Using baseline B and Tfh-like cell counts in SLE patients (Belouski et al., 2010, Cytometry B Clin. Cytom. 78 (1): 49-58) and predicting human cell depletion parameters to simulate human fucosyl h11G2 XC154 / XC155 Depletion kinetics of B and Tfh-like cells after administration.

The predicted human effective dose of fucosyl h11G2 XC154 / XC155 is about 10 to 30 mg (IV), and is based on the assumption that it takes about 8 weeks to deplete B cells in the blood to £ 1 cell / mL to achieve Clinical efficacy. As mentioned above, the fucosyl h11G2 XC154 / XC155 serum concentration after the second administration of 10 to 30 mg IV (administered on days 1 and 29) is estimated, and the predicted C _max is approximately 5 to 15 µg / mL, the predicted area under the concentration-time curve (AUC _tau ) relative to the dosing time interval is about 38 to 114 μg / day / ml, and the predicted average concentration (C _av ; calculated AUC _tau / 28) 1.36 to 4.06 µg / mL.
Example 17: Toxicological study of defucosylated h11G2 XC154 / XC155

In an overview of the toxicity test studies as outlined in Table 14, the defucosylated h11G2 XC154 / XC155 was evaluated in a series of non-clinical toxicity studies. Choose intravenous (IV) or subcutaneous (SC) administration route because it is the required route for clinical administration. As used in this experiment, the IV and SC formulations contained the following components: 50 mg / mL of the CXCR5 antibody or antigen-binding fragment of the invention, 20 mM histidine, 8.5% sucrose, and 0.02% polysorbate 80, 0.005 % EDTA at pH 5.8. As previously discussed elsewhere in this article, studies using human or cynomolgus macaque peripheral blood mononuclear cells indicate that fucosyl h11G2 XC154 / XC155 binds CXCR5 expressing cells with considerable affinity and is similar between human and monkey cells Trigger antibody-dependent cytotoxicity (ADCC). As also previously explained elsewhere herein, the fucosyl h11G2 XC154 / XC155 does not bind to mouse, rat or rabbit CXCR5 heterologs. Therefore, non-clinical toxicity studies are only conducted in cynomolgus monkeys.
Table 14

The abbreviations in Table 14 are as follows: GLP = good laboratory practice; IV = intravenous; OECD = Organization for Economic Cooperation and Development; PBMC = peripheral blood mononuclear cells; SC = subcutaneous.

Conduct all GLP studies in mutually accepted member countries that comply with OECD data. All in vivo studies were conducted with male and female animals. Unless otherwise specified, all doses are expressed as mg protein / kg body weight / dose. Analyze human whole blood (soluble phase) at 1, 10, 100 or 1000 µg / mL or human PBMC (solid phase) at 1, 10 or 100 μg / well.

In IV and SC studies, weekly dosing up to 400 mg / kg / dose (IV) for 17 days (total 3 doses) or every 2 weeks up to 200 mg / kg / dose (IV) for 2 months ( A total of 5 doses were administered) Fucosyl h11G2 XC154 / XC155 was administered to monkeys. The 17-day exploratory monkey study included a recovery phase to day 352. The key (GLP) February monkey study has an 11-month recovery period (day 401). The fucosyl h11G2 XC154 / XC155 induces interleukin release in in vitro analysis. The target organs identified in these studies are blood lymphocyte production systems. The NOAEL in the 2-month monkey study was the highest test dose of 200 mg / kg / dose (IV) on Day 43, with a C _max of 5220 µg / mL and an AUC ₁₆₈ of 438,000 µg • h / mL.
Repeated dose toxicity

The fucosyl h11G2 XC154 / XC155 was used in cynomolgus monkeys for exploratory and key repeated dose toxicity studies.
Exploratory toxicity study

In exploratory non-GLP studies, 0 (IV, SC vehicle), 40 (IV), 260 (SC), or 400 (IV) mg / kg / dose (total 3 doses) by IV or SC injection Fucosyl h11G2 XC154 / XC155 was administered to monkeys (1-2 sex / dose) once a week, followed by the recovery phase in the vehicle, 40 (IV) and 400 (IV) groups (1 / sex / Group) to the 352th day. All animals survived until an autopsy was scheduled on day 17 or 352. There are no clinical signs related to the test article and no effect on body weight, food consumption or serum cytokines.

During the dosing phase, all B cells, CXCR5 + B cells and circulating Tfh-like cells in the peripheral blood relative to the baseline starting on Day 2 or Day 3 (0.00x-0.02x, 0.00x-0.02x and 0.03x, respectively) -0.09x) reduction, the largest reduction from baseline was observed on day 17. At all doses, the magnitude of depletion is similar. In the spleen, significantly reduced numbers of B cells, CXCR5 + B cells, and bona fide Tfh cells were observed at all doses compared to the control (0.018x-0.144x, 0.003x-0.033x, and 0.037x, respectively) -0.245x). In peripheral blood, at ³40 mg / kg / dose, in all animals except one, absolute lymphocytes decreased by day 2 (0.33x-0.67x baseline) and observed at ³40 on day 17 Immunoglobulin (Ig) G in animals increased at mg / kg / dose (1.11x-1.81x baseline). On day 2, there was a brief decrease in natural killer (NK) cell count (0.05x-0.14x baseline), and in most animals, partial to full recovery occurred by day 6. Splenic lymphoid follicles have moderate to significant reduction in cell content; at ³40 mg / kg / dose and 260 mg / kg / dose (SC), there is very little cell content of lymph follicles in the submandibular, axillary and inguinal lymph nodes Reduced, there is microscopic mixed cell infiltration at the injection site. Immunohistochemistry showed a dose-independent reduction of CD20 positive cells and CXCR5 positive cells in the spleen, and decreased CD20 positive cells in the submandibular and inguinal lymph nodes. The effect on lymphoid tissue is related to the reduction of B cells and Tfh-like cells and total lymphocytes in peripheral blood, and to the pharmacology of fucosyl h11G2 XC154 / XC155 (which is a depleting antibody targeting CXCR5 expressing cells) Consistent learning.

During the recovery phase, the decrease in peripheral blood counts of B cells, CXCR5 + B cells, and Tfh-like cells increased to (slowly) day 199, and on day 352, at the end of the recovery phase, all of these cell types in all animals had Partial or complete recovery. During the recovery phase, NK cells and IgG returned to the baseline range (Figure 8A to Figure 8D). There are no microscopic findings related to the test article (including CD20 and CXCR5 immunohistochemical assessment of the spleen and submandibular and inguinal lymph nodes) and in the spleen, the number of B cells, CXCR5 + B cells and bona fide Tfh cells are similar to the vehicle The value of the control animal or higher than it indicates complete recovery.

At any dose, no effect on serum interleukins or T cells, T helper cells or T cytotoxic cells in peripheral blood was observed. No significant anti-drug antibodies (ADA) were detected in this study.

8A-8D show the depletion and recovery of peripheral blood B cells and Tfh-like cells in cynomolgus monkeys. By day 352 of the exploratory toxicity study, blood B cells (Figures 8A and 8B) and Tfh-like cells (Figure 8A and Figure 8B) and Tfh-like cells per microliter of blood in males (Figures 8A and 8C) and females (Figures 8B and 8D) were displayed Figure 8C and Figure 8D) content. B cells are defined as CD3-CD20 +. Tfh-like cells are defined as the sum of CD3 + CD4 + CD95 + CXCR5 + cells and CD3 + CD4 + CD95 + hIgG + cells because the test piece interferes with the CXCR5 antibody used for immunophenotyping. The historical range of B cells in male monkeys (Figure 8A, 272-2503 cells per microliter) and female monkeys (Figure 8B, 233-1700 cells per microliter) is indicated by the dotted line.
Key ( GLP ) toxicity studies

In key studies, the monkeys were given 0 (IV, SC), 5 (IV), 20 (SC), or 200 (IV) mg / kg / dose (total 5 doses) to the monkeys every 2 weeks by IV or SC injection (3-8 mice / sex / group) Fucosyl h11G2 XC154 / XC155 was administered, followed by an 11-month recovery period (day 401). Monkeys were injected with keyhole serocyanin (KLH) and tetanus toxoid (TT) on day 22 (administration phase) and day 253 (recovery phase) to assess the primary and secondary T cell-dependent antibody responses, respectively ( TDAR). Animals were vaccinated with TT but vaccinated with untreated KLH before the start of the study. Before the start of the study (baseline) on Day 22, Day 25, Day 29, Day 36, Day 43, Day 58, Day 253 (before the immunization in the recovery phase), Day 256, Day 260 Blood samples were collected on days, 267 days, 274 days, and 281 days and the production of anti-KLH-IgM, anti-KLH-IgG, anti-TT-IgM, and anti-TT-IgG was evaluated.

Animals survived to the 58th day at all dosing stages and scheduled autopsy. There are no clinical signs related to the test article and no effect on body weight, food consumption, plasma cytokines, coagulation, clinical chemistry, TDAR-KLH parameters or electrocardiogram parameters. In this study, all fucosyl h11G2 XC154 / XC155-related findings include: at 5 and 200 mg / kg / dose IV and 20 mg / kg / dose SC, non-hematological and immunophenotypic parameters in peripheral blood Adverse and reversible changes, TDAR-TT IgG value dose-independent reduction, and microscopic findings and immune phenotype changes of spleen and / or lymph nodes in males and females. During the recovery phase, a control female was euthanized on day 330 due to blood collection complications. All remaining recovered animals survived until necropsy on day 401.

The hematological changes associated with fucosyl h11G2 XC154 / XC155 included a slight to moderate decrease in the overall dose independence of lymphocytes (0.34x-0.60x baseline) at all doses, with the highest incidence on day 2 and subsequent The incidence at the time point is low, and its contribution is that at all doses, the individual animal's total white blood cell count decreases (0.25x-0.59x baseline). Lymphocytes are approximately the baseline and / or control values during the recovery period of the first 1 to 2 months. On day 2 at 200 mg / kg / dose IV and for individual animals at subsequent time points and subsequent recovery within the first 1 to 3 months recovery period, at 5 mg / kg / dose IV and 20 mg / There is also a slightly reduced basophilic sphere (0.17x-0.50x) at kg / dose SC, and the female's red blood cell mass (hemoglobin, red blood cell count, and hematocrit) at 200 mg / kg / dose IV on day 6 ) Briefly decreases slightly (0.79x-0.82x).

As early as the second day, in all animals and dose groups, there was a significant fucosyl h11G2 XC154 / XC155-related reduction in total B cells, CXCR5 + B cells, and Tfh-like cells in peripheral blood relative to baseline (0.00x- 0.56x), and maintained until the end of the dosing phase (day 58), during the recovery phase to day 393, return to baseline or have an absolute value corresponding to each subset, the subset is at 5 mg / kg / Vehicle IV-administered animals in vehicle control animals within the range of values observed (Figures 9A-9D). In all animals, the number of NK cells in peripheral blood on day 2 also decreased significantly from baseline (0.04x-0.46x); however, by day 6 most of the animals were partially to fully recovered, although some animals The NK cells remained below the baseline level at one or more subsequent time points and recovered to the baseline by the end of the recovery phase. In some animals, on day 2, total T cells, T helper cells, and T cytotoxic cells also briefly decreased (0.45x-0.66x); however, by day 6 these cell populations returned in most animals These cell populations in all animals returned to the baseline level by the baseline level and by day 36. At any dose, interleukin (IL) -2, IL-6, IL-10, IL-13, interferon (IFN) -γ and tumor necrosis factor (TNF) -α are absent fucose h11G2 XC154 / XC155 related changes.

At ³5 mg / kg / dose IV and 20 mg / kg / dose SC, splenic lymph follicles, lymphoid follicles in axillary and mesenteric lymph nodes, and cell content of intestinal associated lymphoid tissue (GALT) were observed The dose independence of glycosyl h11G2 XC154 / XC155 is reduced. This is related to the lymphoid follicular cell content of CD20 + and CXCR5 + cells in the spleen assessed by immunohistochemistry, except for one male at 20 mg / kg / dose SC; however, the The increased incidence affects the exposure of fucosyl h11G2 XC154 / XC155 in this animal. In addition, the number of B cells, CXCR5 + B cells, and Tfh cells in the spleen was lower at the 58th autopsy compared to the vehicle control. Some animals have a lower number of NK cells in the spleen than the control. At the end of recovery on Day 401, the absolute value of each subgroup detected in all animals administered fucosyl h11G2 XC154 / XC155 was within the range of the value of the animals in the vehicle control group, indicating that the study gave The lower number of fucosyl h11G2 XC154 / XC155-related recovery observed during the drug phase.

At ³5 mg / kg / dose IV and 20 mg / kg / dose SC, the lymphocyte content of the lymph follicles in the spleen and lymph nodes is reduced and the lower number of B cells, CXCR5 + B cells, Tfh cells and peripheral blood in the spleen The total and total lymphocytes and B cells, CXCR5 + B cells and Tfh-like cells are associated with a decrease. At the end of the recovery phase (day 401), there were no microscopic findings related to fucosyl h11G2 XC154 / XC155 in the 5 mg / kg IV dose group, indicating complete recovery. These findings are consistent with the pharmacology of the fucosyl h11G2 XC154 / XC155 expected to deplete CXCR5 expressing cells. A lower number of NK cells in peripheral blood and / or spleen can be attributed to NK effector cell death (which has been described in NK cells that follow antibody-dependent cytotoxicity (Warren et al., 2011, J. Immunol. Meth. 370: 86-92)) and / or tissue redistribution; however, other mechanisms are not excluded.

There is no fucosyl h11G2 XC154 / XC155-related effect on the initial T cell-dependent antibody response (TDAR) of keyhole snail hemocyanin (KLH). Anti-KLH-immunoglobulin (Ig) M and IgG responses were observed in all groups. Prior to the study, all monkeys were vaccinated against tetanus toxoid (TT). Increased IgM and IgG memory responses were observed in all animals in all dose groups. Seven days, 14, 21, and 36 days after immunization, secondary TDAR (IgG to TT) was observed in male and female animals in the 5 IV, 20 SC, and / or 200 IV mg / kg / dose groups. The central point effect value) of h11G2 XC154 / XC155 related dose independence. At 7 days after immunization, a statistically significant reduction in the average anti-TT IgG antibody was observed for the 5 and 200 mg / kg / dose IV groups.

After immunization during the recovery phase, anti-KLH IgM and IgG were present in all animals in the 5 mg / kg fucosyl h11G2 XC154 / XC155 dose group at one or more time points within the vehicle control range Memory response. During the recovery time point, 7 days after the immunization in the recovery phase, a group of anti-TT-IgG central point titers (CPT) values observed in the 5 mg / kg dose group of animals had the presence of fucosyl h11G2 XC154 / The statistics related to XC155 decreased significantly. The CPT values of all animals in the recovery phase were within the range of the control group, except for 2 of the 8 animals in the 5 mg / kg dose group. These animals were at all recovery time points until 28 days after the TT immunization in the recovery phase All have CPT values lower than the control range. There was no change in the anti-TT IgM value, which was attributed to the administration of fucosyl h11G2 XC154 / XC155 during the administration or recovery phase.

There were initial anti-KLH IgM and IgG responses in all groups; at one or more time points after the initial immunization, some animals in the 5 and 200 mg / kg / dose groups had CPTs higher than the control range. At any time point, there was no statistically significant difference in the average anti-KLH IgM or IgG antibodies in either group. Because individual animal changes are sporadic, lack of dose dependence, and group CPT values are within the range of control animals, it is not considered to be related to fucosyl h11G2 XC154 / XC155. During the recovery time point, all animals were within the control range, except for 1 out of 8 animals in the 5 mg / kg dose group, which had slightly lower than the control range at 14, 21, and 28 days after KLH immunization CPT value. These data (dose or recovery phase) indicate that all animals (males and females) administered fucosyl h11G2 XC154 / XC155 are able to produce initial IgM and IgG responses to KLH vaccination similar to control animals.

Although as detected by immunophenotyping, total B cells, CXCR5 + B cells, Tfh-like cells and NK cells in peripheral blood were significantly reduced and the number of B cells, CXCR5 + B cells and Tfh cells in the spleen were significantly lower, the lymphocyte count Decreased and decreased lymphocyte content in the spleen, lymph nodes, and GALT, but all findings are not harmful, due to lack of relevant clinical signs and lack of impact on initial TDAR. Other blood findings include reductions in the parameters of total white blood cells, basophiles, and red blood cells, which are not harmful, due to the limited severity and lack of microscopic correlation. No microscopic and immunohistochemical findings related to fucosyl h11G2 XC154 / XC155 were observed on day 401.

After SC or IV administration every other week for 2 months, the NOAEL of fucosyl h11G2 XC154 / XC155 in monkeys was 200 mg / kg / dose. Under NOAEL, the systemic exposure (C _max and AUC ₁₆₈ ) were 5220 µg / mL and 438,000 µg • h / mL, respectively. Anti-fucosyl h11G2 XC154 / XC155 antibodies were detected in this study and the data are summarized elsewhere in this article. The risk assessment of target organ toxicity observed in repeated dose toxicity procedures is provided elsewhere in this article. The threshold concentration of fucosyl h11G2 XC154 / XC155 related to key reactions in monkeys can be found elsewhere in this article.
Reproductive and developmental toxicity

In cynomolgus macaques, there were no test article-related effects in male or female reproductive tissues evaluated in repeated dose toxicity studies.
Local tolerance

Despite the macroscopic and microscopic examination of the injection site in in vivo toxicity studies, the fucosyl h11G2 XC154 / XC155 has not been used for independent local tolerance studies.

In the exploratory monkey study, there was very little to slight infiltration at the SC injection site compared to control animals. At the SC injection site at 260 mg / kg / dose, there was moderate perivascular mixed leukocyte infiltration (mononuclear leukocytes) , Neutrophils and less eosinophils). In 260 mg / kg / dose SC males, infiltration of the multi-focal lesions into the sarcolemma of small blood vessels. In SC females of 260 mg / kg / dose, several multinucleated giant cells infiltrate the subcutaneous collagen, and there are intracytoplasmic basophilic or eosinophilic small pieces of fibrous material in these cells. In the key 2-month monkey study, the microscopic findings of IV and SC administration sites were not considered to be related to the test items.
Antigenicity

The immunogenicity was assessed by measuring the ADA level in repeated dose toxicity studies in cynomolgus cynomolgus monkeys; this data was previously described in this article.
Immunotoxicity

In vitro and in vivo studies characterized the effect of fucosyl h11G2 XC154 / XC155 on the immune system. In human cytokinin release analysis, two different in vitro types were used to determine the possibility of fucosyl h11G2 XC154 / XC155-induced cytokines (TNF-a, IL-6 and IFN-g) release: using whole blood Soluble phase analysis and solid phase analysis using fixed fucosyl h11G2 XC154 / XC155 and peripheral blood mononuclear cells (PBMC). Samples from 8 healthy human donors were evaluated in each of these types. In both types of cytokine release analysis, cytokine release (TNF-a, IL-6 and IFN-g) induced by h11G2 XC154 / XC155 was observed. In vivo, repeated dose exploratory and key toxicity studies in monkeys characterize the in vivo cytokine release profile and the effect of fucosyl h11G2 XC154 / XC155 on immune system cells and tissues. Fucosyl h11G2 XC154 / XC155 did not induce cytokines in vivo. As described elsewhere in this article, depletion of lymphocytes, B cells, CXR5 + B cells, and Tfh / Tfh-like cells was observed in the peripheral blood and spleen of monkeys administered fucosyl h11G2 XC154 / XC155. The expected mechanism of action of CXCR5 depleting antibodies is the same. After the recovery phase in exploratory and key toxicity studies, the lymphocyte parameters in the spleen and lymph nodes were comparable to vehicle controls.

The initial and secondary TDAR responses to KLH and TT were evaluated in key 2-month toxicity studies as discussed elsewhere in this article. There is no fucosyl h11G2 XC154 / XC155 related effect on the initial TDAR of KLH. All animals developed a secondary TDAR response to TT, but during the dosing and recovery phase of the study, 7 days after immunization, 5 and 200 mg / kg / dose of the central point titer of Group IV were observed The glycosyl h11G2 XC154 / XC155-related reduction.
Tissue cross-reactivity ( TCR )

Extensive preliminary method development research was carried out in the preliminary dyeing method research using fucosyl h11G2 XC154 / XC155, including multiple tests and methods. None of these analysis cases consistently indicated that the expected cells and tissues had membrane staining. In a GLP-compliant tissue cross-reactivity study, assess the biotin-labeled fucosyl h11G2 XC154 / XC155 (fucosyl h11G2 XC154 / XC155-Bio; 1 and 5 mg / mL) and cynomolgus monkeys and human tissues Combine frozen sections. The stained pattern overlaps between the cynomolgus macaque and human tissue. The common staining of humans and monkeys was observed in monocytes, reticuloendothelial cells, various epithelial cells, glial cells and / or pituitary cells, indicating the expected fucosyl h11G2 XC154 / XC155 reactivity (Breitfeld et al., 2000 , J Exp Med 192 (11): 1545-1552; Carlsen et al., 2002, Gut 51 (3): 364-371; Flynn et al., 2003, J. Neuroimmunol. 136 (1-2): 84-93; Schaerli et al., 2000, J. Exp. Med. 192 (11): 1553-1562; Schmutz et al., 2005, Arthritis Res. Ther. 7 (2): R217-R229), and colloids of the thyroid were also observed. For other monkey or human tissues, positive fucosyl h11G2 XC154 / XC155 staining was observed. For human tissues, staining of nerve fibers in the optic nerve and neurons in the spinal nerve roots, smooth muscle cells in the prostate, interstitial cells in the testis, and crystalline fibers in the eye was observed.

The tissues stained only in cynomolgus macaques are hematopoietic precursor cells in bone marrow, mast cells in skin and cervix, nerve fiber nets in brain and spinal cord, cardiomyocytes and spindle cells in placenta and optic nerve sheath. Although this may represent an unexpected cross-reaction of the test article, the staining occurs essentially in the cytoplasm, does not have microanatomic specificity, and lacks clear plasma membrane staining. This type of cytoplasmic staining is not good at distinguishing CXCR5 expressing cells and tissues from CXCR5 expressing cells and tissues. Cytoplasmic staining is not expected to enter the test article in vivo and is generally considered to have little or no toxicological significance (Hall et al., 2008, Preclinical Safety Evaluation of Biopharmaceuticals: A Science-Based Approach to Facilitating Clinical Trials, pages 208-240 , Cavagnaro, edited by JA, Wiley-Interscience; Leach et al., 2010, Toxicol. Pathol. 38 (7): 1138-1166). The lack of clear membrane staining indicates that a reliable method cannot be obtained for a complete evaluation. No adverse findings in the 2-month repeated dose toxicity study indicate that the staining observed in the in vitro tissue cross-reactivity study did not translate into in vivo effects. Despite repeated attempts to obtain a reliable TCR method, the observed combination did not achieve safety considerations beyond expectations based on the required pharmacology.
The relationship between discovery and pharmacokinetics

After administration of 5 (IV), 20 (SC) or 200 (IV) mg / kg / dose to SC and IV of male and female monkeys every 2 weeks for 2 months (a total of 5 doses), the fucose was measured Serum concentration of H11G2 XC154 / XC155. In all dose groups, there was no significant gender-related difference in systemic exposure; therefore, the combined data of male and female cynomolgus monkeys were used to present the average TK parameters of the group. The systemic exposure was higher after repeated administrations, with the cumulative ratio (Study Day 43 / Study Day 1) in the range of approximately 1.4 to 1.7. In addition, systemic exposure increased in a dose proportional manner after IV administration. After SC administration, the bioavailability of fucosyl h11G2 XC154 / XC155 was estimated to be> 50%.

The threshold serum concentrations of fucosyl h11G2 XC154 / XC155 related to the critical response and exposure limit calculated for these critical reactions are provided in Table 15.
Table 15

The abbreviations used in Table 15 are as follows: AUC = area under the concentration-time curve; C _av = average concentration; C _max = maximum (average) plasma concentration; Tfh-like cells (alternatively cTfh); CXCR5 = chemotaxis Receptor type 5; GALT = gut-associated lymphoid tissue; IgG = immunoglobulin G; IV = intravenous; NK = natural killer; NOAEL = no visible harmful effects level; SC = subcutaneous; TDAR = T cell-dependent antibody response ; Tfh = follicular T helper cells; TT = tetanus toxoid; WBC = white blood cells.

In repeated dose studies, the C _max value indicates the average plasma concentration. The _Cav value is calculated by dividing the AUC by the sampling interval (48 or 168 hours). The reported value is obtained near the end of the dosing phase.

The exposure limit is calculated by dividing the C _av value in animal toxicity studies by the predicted 30 mg, the IV human effective dose of 4.06 mg / mL human C _av .
Target organ toxicity

Based on the non-clinical studies conducted, the blood lymphocyte production system (spleen, lymph nodes, GALT, tonsils, circulating lymphocytes, white blood cells, red blood cell parameters, in vitro cytokine release) was identified as a potential target organ / tissue. In a non-critical 17-day monkey study using fucosyl h11G2 XC154 / XC155, the changes related to the test article in these tissues were consistent with those observed in the key February toxicity study. In non-critical and critical toxicity studies, all fucosyl h11G2 XC154 / XC155 related findings returned fully or partially to baseline or vehicle control values during the recovery phase. None of the test article-related effects is considered harmful, due to the lack of any clinical findings or opportunistic infections, and to the minimal impact on immune function measured by TDAR analysis. Therefore, a NOAEL of 200 mg / kg / dose IV in monkeys was established for fucose-based h11G2 XC154 / XC155 related effects.
Vascular lymphocyte generating system

In repeated dose toxicity studies in cynomolgus monkeys, starting from the second study day, peripheral blood lymphocytes and rocks were observed at doses of ³5 mg / kg / dose (IV) and 20 mg / kg / dose (SC) The reduction of trehalose-based h11G2 XC154 / XC155. These reductions are related to the observed reductions in total B cells, CXCR5 + B cells, and Tfh-like cells, which are maintained until day 57. The reduction of these lymphocyte subpopulations to the end of the recovery phase in the exploratory toxicity study showed a complete or partial recovery to baseline values. On the second day, there was also a reduction in fucose-based h11G2 XC154 / XC155 related to NK cells, total T cells, T helper cells and T cytotoxic cells at ³5 mg / kg / dose, but by the time of administration End the realization part to fully return to the baseline value. In addition to the decrease in the population of lymphocytes in the peripheral blood, a very brief decrease in RBC mass was observed on day 6 for female monkeys dosed with 200 mg / kg / dose.

The decrease in the population of lymphocytes in the peripheral blood is related to the lower lymphocyte content associated with fucosyl h11G2 XC154 / XC155 in the lower spleen and lymph nodes of ³5 mg / kg / dose. A lower number of total B cells, CXCR5 + B cells, bona fide Tfh cells, and NK cells in the spleen, as well as lower follicular CD20 + and CXCR5 + cells, responded to lower lymphocyte content. Lower levels of follicular lymphocytes were observed in lymph nodes and GALT at ³5 mg / kg / dose and tonsils at ³40 mg / kg / dose.

After the recovery phase in exploratory and key toxicity studies, the lymphocyte parameters in the spleen and lymph nodes were comparable to vehicle controls.

The reduction of total B cells, CXCR5 + B cells and Tfh / Tfh-like cells is consistent with the expected mechanism of action of fucosyl h11G2 XC154 / XC155, which is expected to deplete CXCR5 expressing cells. The lower number of NK cells in the peripheral blood and spleen may be due to effector cell death known to follow ADCC in NK cells.

Although a decrease in lymphocyte parameters was observed in the peripheral blood and spleen of monkeys administered fucosyl h11G2 XC154 / XC155, there was no related effect on fucosyl h11G2 XC154 / XC155 in the initial TDAR of KLH. All animals produced a second TDAR for TT, but a statistically significant reduction was observed at a dose of ³5 mg / kg / dose.

It is believed that all fucosyl h11G2 XC154 / XC155-related findings are harmless, which is attributed to the lack of relevant clinical signs or opportunistic infections. The effect of fucosyl h11G2 XC154 / XC155 on peripheral blood lymphocytes, total B cells, CXCR5 + B cells, Tfh-like cells and NK cells, as well as the initial and secondary TDAR response can be monitored in the human body in the clinic.

In soluble and solid in vitro cytokine release analysis formats, fucoyl h11G2 XC154 / XC155-induced cytokine release (TNF-a, IL-6 and IFN-γ) was observed. Due to the expected pharmacology of this molecule, the fucosyl h11G2 XC154 / XC155-induced interleukin release is expected in vitro. In exploratory or key in vivo monkey studies, the fucosyl h11G2 XC154 / XC155 did not induce cytokine release. It is easy to monitor the serum cytokines and clinical indicators of systemic cytokine release in the clinic.
Table 16 (Sequence)
(CDR amino acid residues are underlined)

Although the teachings disclosed have been described with reference to different applications, methods, kits, and composition descriptions, it will be understood that various changes and modifications can be made without departing from the teachings herein and the invention claimed below. The foregoing examples are provided to better illustrate the teachings disclosed and are not intended to limit the scope of the teachings presented herein. Although the teachings of the present invention have been described in terms of these exemplary embodiments, skilled artisans will readily understand that many variations and modifications of these exemplary embodiments are possible without undue experimentation. All such changes and modifications are within the scope of the current teaching.

All references cited in this article (including patents, patent applications, papers, textbooks, and the like) and references cited therein are incorporated by reference in their entirety to the extent that they have not yet been cited. purpose. In the event that one or more of the incorporated documents and similar materials (including but not limited to defined terms, term usage, described technology, or the like) is different or inconsistent with this application, the application is regarded as quasi.

It will be apparent to those skilled in the art that various modifications and changes can be made in the present invention without departing from the scope or spirit of the invention. Considering the description and practice of the present invention disclosed herein, those skilled in the art will understand other embodiments of the present invention. It is only hoped that the description and examples are regarded as illustrative, wherein the true scope and spirit of the present invention are indicated by the following patent application scope.

When reading in conjunction with the accompanying drawings, the foregoing summary of the invention and the following embodiments will be better understood. To illustrate the invention, it is shown in the illustrated embodiment. However, it should be understood that the invention is not limited to the precise arrangements and instrumentalities shown.

FIG. 1 is a diagram illustrating a typical two-antennary glycoform commonly found in the constant region of immunoglobulin heavy chains. This diagram shows the Eu numbering according to Edelman et al., 1969, Proc. Natl. Acad. Sci. USA 63 (1): 78-85, 1991, as described by Kabat et al., At amino acid residue number 297 The glycoform of asparagine linkage (N-linkage) on the constant domain of IgG (asparagine 297; Asn297 ).

Figures 2A - 2G are diagrams depicting exemplary two-antennary glycoforms commonly found in the constant region of immunoglobulin heavy chains. "G0" means a two-antennary structure in which no terminal sialic acid (NeuAc) or Gal exists, "G1" means a two-antennary structure with one Gal and no NeuAc and "G2" means a two-antennary structure with no GalAc and no NeuAc The second antenna structure. In each glycoform, fucose is usually present in antibodies produced in mammalian cells, such as CHO cells. Figure 2A shows G2S2, Figure 2B shows G2S1, Figure 2C shows G2, Figure 2D shows G1, Figure 2E shows G0, Figure 2F shows G-1, and Figure 2G shows G-2.

Figure 3 depicts a bar graph showing various monoclonal antibodies (represented on the x-axis) and HEK 293 cells (brighter bars) expressing human CXCR5 (hCXCR5 293) and / or HEK expressing cynomolgus monkey CXCR5 (cynoCXCR5 293) 293 cells (darker bars) binding.

Figure 4A depicts a diagram showing the detectable binding of various anti-CXCR5 antibodies to cells expressing CXCR1.

Figure 4B depicts a diagram showing the lack of binding of various anti-CXCR5 antibodies to cells expressing CXCR2.

Figure 4C depicts a diagram showing the lack of binding of various anti-CXCR5 antibodies to cells expressing CXCR3.

Figure 4D depicts a diagram showing the binding of various anti-CXCR5 antibodies to Jurkat cells that naturally express CXCR4.

Figure 5 depicts a graph showing the binding affinity of antibodies to CXCR5 expressing cells: defucosylated h11G2 XC154 / XC155 (open circles), fucosylated h11G2 XC154 / XC155 (filled circles), 2C9 (filled triangles), 16D7 (solid square) and 11A7 (solid oval).

Figure 6 shows the alignment of human and mouse CXCR5 amino acid sequences. The figure also shows the various extracellular domains of CXCR5 labeled "N", "L1", "L2" and "L3" underlined.

Figure 7 depicts a bar graph showing that certain amino acid residues are essential for antibodies and hCXCR5. That is, changing D10G or inserting SI into humans sequenced to eliminate the binding of antibody 2C9 but did not affect the binding of the other three antibodies. More importantly, replacing L11T or D22A eliminates the binding of 11G2 but does not affect the binding of 2C9, 16D7 or 11A7. Changing W to K (W19K) at amino acid residue number 19 eliminated the binding of antibodies 16D7 and 11A7 but did not affect the binding of 11G2 and 2C9. These data confirm that these four antibodies do not share the same epitope on hCXCR5.

Figure 8A depicts a graph showing the depletion and recovery of peripheral blood B cells in male cynomolgus macaques. A 352-day exploratory toxicity study showed the number of peripheral blood B cells per microliter of blood in males. B cells are defined as CD3-CD20 +. Display individual animal information. The historical range of B cells in male monkeys (272-2503 cells / microliter) is indicated by a dashed line.

Figure 8B depicts shows depletion and recovery of peripheral blood B cells in female cynomolgus monkeys than in FIG. A 352-day exploratory toxicity study showed the number of B cells in the peripheral blood per microliter of blood in females. B cells are defined as CD3-CD20 +. Display individual animal information. The historical range of B cells in female monkeys (233-1700 cells / microliter) is indicated by a dashed line.

Figure 8C depicts a graph showing the depletion and recovery of Tfh-like (also known as "cTfh" or "circulating Tfh" cells) cells in male cynomolgus monkeys. A 352-day exploratory toxicity study showed the number of Tfh-like cells in the peripheral blood per microliter of blood in males. Tfh-like cells are defined as the sum of CD3 + CD4 + CD95 + CXCR5 + cells and CD3 + CD4 + CD95 + hIgG + cells because the test piece interferes with the CXCR5 antibody used for immunophenotyping. Display individual animal information.

Figure 8D shows depicting female cynomolgus macaques Tfh kind (also known as "cTfh" or "circulation Tfh" cells) of cell depletion and recovery of the map. A 352-day exploratory toxicity study showed the number of Tfh-like cells in the peripheral blood per microliter of blood in females. Tfh-like cells are defined as the sum of CD3 + CD4 + CD95 + CXCR5 + cells and CD3 + CD4 + CD95 + hIgG + cells because the test piece interferes with the CXCR5 antibody used for immunophenotyping. Display individual animal information.

9A depicts depletion and recovery of peripheral blood B cells in cynomolgus monkeys show other than FIG. A 393-day key GLP toxicity study showed the number of peripheral blood B cells per microliter of blood in monkeys. B cells are defined as CD3-CD20 + HLA-DR + cells. Display group average data (male and female combination) +/- standard deviation.

Figure 9B depicts in addition shows cynomolgus CXCR5 + peripheral blood B cell depletion and recovery of FIG. A 393-day key GLP toxicity study showed the number of CXCR5 + B cells in peripheral blood per microliter of blood in monkeys. B cells are defined as CD3-CD20 + HLA-DR + cells. Display group average data (male and female combination) +/- standard deviation.

Figure 9C depicts a graph showing the depletion and recovery of peripheral blood circulating follicular T helper cells (cTfh; also referred to elsewhere as "Tfh-like cells") in cynomolgus monkeys. A 393-day key GLP toxicity study showed the number of peripheral blood cTfh cells per microliter of blood in monkeys. cTfh cells are defined as CD3 + CD4 + CD95 + cells. Display group average data (male and female combination) +/- standard deviation.

Figure 9D depicts depletion of peripheral blood cTfh CXCR5 + cells (described elsewhere also known as "Tfh like cells") and the restoration of the map to show detectable surface CXCR5 outside cynomolgus monkeys have. A 393-day key GLP toxicity study showed the number of CXCR5 + cTfh cells in peripheral blood per microliter of blood in monkeys. cTfh cells are defined as CD3 + CD4 + CD95 + cells. Display group average data (male and female combination) +/- standard deviation.

Claims (28)

An isolated antibody that specifically binds CXCR5, or an antigen-binding fragment thereof, wherein the anti-system is at least one antibody selected from the group consisting of: a) Antibodies comprising: CDR-L1 comprising amino acid sequence SEQ ID NO: 2; CDR-L2 comprising amino acid sequence SEQ ID NO: 3; CDR-L2 comprising amino acid sequence SEQ ID NO: 4 CDR-L3; CDR-H1 comprising the amino acid sequence SEQ ID NO: 7; CDR-H2 comprising the amino acid sequence SEQ ID NO: 8; and CDR-H3 comprising the amino acid sequence SEQ ID NO: 9; b) Antibodies comprising: CDR-L1 comprising the amino acid sequence SEQ ID NO: 2; CDR-L2 comprising the amino acid sequence SEQ ID NO: 3; CDR-L2 comprising the amino acid sequence SEQ ID NO: 4 CDR-L3; CDR-H1 comprising the amino acid sequence SEQ ID NO: 7; CDR-H2 comprising the amino acid sequence SEQ ID NO: 8; and CDR-H3 comprising the amino acid sequence SEQ ID NO: 11; c) Antibodies comprising: CDR-L1 comprising the amino acid sequence SEQ ID NO: 14; CDR-L2 comprising the amino acid sequence SEQ ID NO: 15; CDR-L2 comprising the amino acid sequence SEQ ID NO: 16 CDR-L3; CDR-H1 comprising the amino acid sequence SEQ ID NO: 19; CDR-H2 comprising the amino acid sequence SEQ ID NO: 20; and CDR-H3 comprising the amino acid sequence SEQ ID NO: 21; d) Antibodies containing the following: VL containing the amino acid sequence encoded by the insert deposited with the ATCC and having the accession number PTA-124324 and containing the VL containing the amino acid sequence deposited with the ATCC and having the accession number PTA-124323 VH of the amino acid sequence encoded by the body insert; e) Antibodies comprising: VL comprising the amino acid sequence SEQ ID NO: 1 and VH comprising the amino acid sequence SEQ ID NO: 6; f) an antibody comprising: VL comprising the amino acid sequence SEQ ID NO: 13 and VH comprising the amino acid sequence SEQ ID NO: 18; g) an antibody comprising: VL comprising the amino acid sequence SEQ ID NO: 47, and VH comprising the amino acid sequence SEQ ID NO: 52; h) an antibody comprising: VL comprising the amino acid sequence SEQ ID NO: 5 and VH comprising the amino acid sequence SEQ ID NO: 6; i) an antibody comprising: VL comprising the amino acid sequence SEQ ID NO: 5 and VH comprising the amino acid sequence SEQ ID NO: 10; j) an antibody comprising: VL comprising the amino acid sequence SEQ ID NO: 13 and VH comprising the amino acid sequence SEQ ID NO: 17; k) Antibodies comprising: VL comprising the amino acid sequence SEQ ID NO: 1 and VH comprising the amino acid sequence SEQ ID NO: 12; l) Antibodies comprising: LC comprising the amino acid sequence SEQ ID NO: 22, and HC comprising the amino acid sequence SEQ ID NO: 23; m) an antibody comprising: an LC comprising the amino acid sequence SEQ ID NO: 24, and an HC comprising the amino acid sequence SEQ ID NO: 25; n) Antibodies comprising: LC comprising the amino acid sequence SEQ ID NO: 26, and HC comprising the amino acid sequence SEQ ID NO: 27; o) Antibodies comprising: LC comprising the amino acid sequence SEQ ID NO: 28, and HC comprising the amino acid sequence SEQ ID NO: 29; p) antibodies comprising: VL encoded by the nucleic acid sequence SEQ ID NO: 95, and VH encoded by the nucleic acid sequence SEQ ID NO: 96; and q) Antibodies comprising: LC encoded by nucleic acid sequence SEQ ID NO: 97, and HC encoded by nucleic acid sequence SEQ ID NO: 98. An isolated antibody or antigen-binding fragment thereof that specifically binds to C-X-C-chemokine receptor 5 (CXCR5), wherein the antibody or antigen-binding fragment thereof is at least one antibody selected from the group consisting of: a) The following antibody or antigen-binding fragment thereof: which binds to the hCXCR5 epitope containing the leucine acid at the amino acid residue number 11 according to the amino acid sequence SEQ ID NO: 32, but does not bind to this residue The base is not the epitope of leucine; b) The following antibody or antigen-binding fragment thereof: which binds to the hCXCR5 epitope comprising leucine at amino acid residue number 11 according to the amino acid sequence SEQ ID NO: 32, but does not bind to it The residue is the epitope of threonine; c) The following antibody or antigen-binding fragment thereof: which binds to the hCXCR5 epitope containing aspartic acid at amino acid residue number 22 according to the amino acid sequence SEQ ID NO: 32, but does not bind to it The residue is not the epitope of aspartic acid; d) The following antibody or antigen-binding fragment thereof: which binds to the hCXCR5 epitope containing aspartic acid at amino acid residue number 22 according to the amino acid sequence SEQ ID NO: 32, but does not bind to it The residue is the epitope of alanine; e) The following antibody or antigen-binding fragment thereof: the binding comprises leucine according to the amino acid sequence SEQ ID NO: 32 at the amino acid residue number 11 and at the amino acid residue number 22 The hCXCR5 epitope of aspartic acid, but does not bind to the epitope in which the leucine is substituted with threonine and / or the aspartic acid is substituted with alanine; f) The following antibody or antigen-binding fragment thereof: which binds to hCXCR5 or a fragment thereof comprising leucine at amino acid residue number 11 according to the amino acid sequence SEQ ID NO: 32, but does not bind to it The residue is not hCXCR5 of leucine or a fragment thereof; g) The following antibody or antigen-binding fragment thereof: which binds to hCXCR5 or a fragment thereof comprising leucine at amino acid residue number 11 according to the amino acid sequence SEQ ID NO: 32, but does not bind to it The residue is hCXCR5 of threonine or a fragment thereof; h) The following antibody or antigen-binding fragment thereof: which binds hCXCR5 or a fragment thereof comprising aspartic acid at amino acid residue number 22 according to the amino acid sequence SEQ ID NO: 32, but does not bind to it The residue is not hCXCR5 of aspartic acid or a fragment thereof; i) The following antibody or antigen-binding fragment thereof: which binds to hCXCR5 or a fragment thereof comprising aspartic acid at amino acid residue number 22 according to the amino acid sequence SEQ ID NO: 32, but does not bind to it The residue is hCXCR5 of alanine or a fragment thereof; and j) The following antibody or antigen-binding fragment thereof: the binding comprises leucine at amino acid residue number 11 according to the amino acid sequence SEQ ID NO: 32 and at amino acid residue number 22 HCXCR5 of aspartic acid or a fragment thereof, but not hCXCR5 or a fragment thereof in which the leucine is substituted with threonine and / or the aspartic acid is substituted with alanine. An isolated antibody or antigen-binding fragment thereof that specifically binds to C-X-C-chemokine receptor 5 (CXCR5), wherein the antibody or antigen-binding fragment thereof is at least one antibody selected from the group consisting of: a) The following antibody or antigen-binding fragment thereof: It binds to hCXCR5 expressed on human B cells with an apparent affinity of EC50 of about 6.60 pM and a standard deviation of about ± 2.33 pM; b) The following antibody or antigen-binding fragment thereof: It binds to hCXCR5 expressed on human circulating follicular T helper-like cells with an apparent affinity of EC50 of approximately 5.89 pM and a standard deviation of approximately ± 1.40 pM; c) The following antibody or antigen-binding fragment thereof: It binds to hCXCR5 expressed on human follicular T helper (Tfh) cells with an apparent affinity of EC50 of about 10.6 pM; d) The following antibody or antigen-binding fragment thereof, which binds cynoCXCR5 expressed on cynomolgus macaque B cells with an apparent affinity of EC50 of about 1.32 pM; e) The following antibody or antigen-binding fragment thereof: It binds to cynoCXCR5 expressed on cynomolgus monkey Tfh-like cells with an apparent affinity of EC50 of about 10.5 pM; f) the following antibody or antigen-binding fragment thereof: it antagonizes CXCR5-CXCL13 signaling in cAMP report analysis with an EC50 of approximately 961 pM; g) The following antibody or antigen-binding fragment thereof: It exhibits ADCC activity on human B cells expressing hCXCR5 with an EC50 of about 2.01 pM and a standard deviation of about ± 2.28 pM; h) The following antibody or antigen-binding fragment thereof: It exhibits ADCC activity on human Tfh-like cells expressing hCXCR5 with an EC50 of about 4.28 pM and a standard deviation of about ± 2.88 pM; i) The following antibody or antigen-binding fragment thereof: which exhibits ADCC activity of human Tfh cells expressing hCXCR5 with an EC50 of about 0.11 pM; j) The following antibody or antigen-binding fragment thereof: which exhibits ADCC activity of cynoCXCR5 expressing cynoCXCR5 cynomolgus macaque B cells with an EC50 of about 15.3 pM and a standard deviation of about ± 11.7 pM; k) The following antibody or antigen-binding fragment thereof: which binds hCXCR5 but does not detectably bind to the human chemotactic receptor CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CMKLR1 CXCR3R1, CXCR1, CXCR2, CXCR3, CXCR4, CXCR6, CXCR7 and XCR1; l) Antibodies or antigen-binding fragments that inhibit the binding of CXCR5 to CXCL13; m) Antibodies or antigen-binding fragments thereof that bind to CXCR5 + human B cells with an apparent affinity of EC50 less than about 26 pM, but not cells expressing CXCR5 mouse, rat, or rabbit heterologs; n) Antibodies or antigen-binding fragments that antagonize the inhibition of cXCL13 released by cAMP triggered by forskolin; o) antibodies or antigen-binding fragments that trigger ADCC of CXCR5 expressing cells in human donor and cynomolgus monkey PBMC and human donor TMC; p) The following antibodies or antigen-binding fragments: which bind to human CXCR5 but not to human chemokine receptors CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CMKLR1, CXCR3R1, CXCR1 CXCR2, CXCR3, CXCR4, CXCR6, CXCR7 or XCR1; q) antibodies or antigen-binding fragments that deplete B cells in peripheral blood; r) antibodies or antigen-binding fragments that deplete Tfh-like cells in peripheral blood; s) antibodies or antigen-binding fragments that deplete bona fide Tfh cells in the spleen; and t) Antibodies or antigen-binding fragments that impair the humoral immune memory response. The antibody of any one of claims 1 to 3, wherein the antibody or antigen-binding fragment thereof exhibits at least one of the following biological activities: a) Binding CXCR5 + human B cells with an apparent affinity of EC50 less than about 26 pM, but not cells expressing CXCR5 mouse, rat, or rabbit xenologs; b) antagonize the inhibition of CXCL13 triggered by forskolin on cAMP release; c) Trigger ADCC of CXCR5 expressing cells in human donor and cynomolgus monkey PBMC and human donor TMC; d) Binding to human CXCR5 but not to human chemokine receptors CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CCR10, CMKLR1, CXCR3R1, CXCR1, CXCR2, CXCR3, CXCR4, CXCR6, CXCR7 Or XCR1; e) Depletion of B cells in the peripheral blood; f) deplete Tfh-like cells in the peripheral blood; g) depletion of bona fide Tfh cells in the spleen; or h) Impair humoral immune memory response. The antibody of any one of claims 1 to 4, wherein the antibody or antigen-binding fragment thereof is defucosylated. An isolated nucleic acid encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5. An isolated nucleic acid encoding VH, VL, or both of an antibody or antigen-binding fragment that specifically binds CXCR5, wherein the nucleic acid comprises: nucleic acid sequence SEQ ID NO: 95, nucleic acid sequence SEQ ID NO: 96, or both. An isolated nucleic acid encoding the heavy chain, light chain, or both of an antibody or antigen-binding fragment that specifically binds CXCR5, wherein the nucleic acid comprises: nucleic acid sequence SEQ ID NO: 97, nucleic acid sequence SEQ ID NO: 98 or both By. An isolated nucleic acid encoding VH, VL, or both of an antibody or antigen-binding fragment that specifically binds CXCR5, wherein the nucleic acid contains the nucleic acid sequence of an insert deposited with ATCC and having a plastid with registration number PTA-124323 The nucleic acid sequence of an insert deposited with ATCC and having a plastid with accession number PTA-124324, or both. A vector comprising the nucleic acid according to any one of claims 6 to 9. A host cell comprising the vector according to claim 10. The host cell according to claim 11, wherein the host cell line is selected from the group consisting of mammalian cells consisting of CHO cells, COS cells, HEK-293 cells, NS0 cells, PER.C6® cells or Sp2.0 cells. The host cell of claim 12, wherein the cell lacks functional α-1,6-fucosyltransferase (FUT8). The host cell according to claim 13, wherein the cell is a Potelligent® CHOK1SV cell or a Lec13 CHO cell. A method for preparing an antibody, or an antigen-binding fragment thereof, under conditions in which an antibody or antigen-binding fragment thereof is expressed by Potelligent® CHOK1SV cells as in claim 14 and is defucosylated, which comprises the host cell. The method of claim 15, further comprising isolating the antibody or antigen-binding fragment thereof. The defucosylated antibody of claim 15, or an antigen-binding fragment thereof, wherein the antibody exhibits enhanced ADCC activity when compared to an antibody or antigen-binding fragment thereof that is otherwise fucosylated. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 and 17, and a pharmaceutically acceptable carrier or excipient. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 and 17, and the pharmaceutical composition according to claim 18, which are used for the treatment of immune diseases, disorders or conditions. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 or 17 or a pharmaceutical composition according to claim 18 for the treatment of immune diseases, disorders or conditions. A method for treating or preventing a CXCR5-mediated immune disease, disorder or condition in a human individual in need, the method comprising administering to the individual an effective amount of a pharmaceutical composition according to claim 18, wherein the disease , Disease or pathology is selected from the group consisting of: inflammatory reactions, such as inflammatory skin diseases, including psoriasis and dermatitis (eg atopic dermatitis); dermatomyositis; systemic scleroderma and sclerosis; and inflammation Sexual bowel disease-related reactions (such as Crohn's disease and ulcerative colitis); respiratory distress syndrome (including adult respiratory distress syndrome; ARDS); dermatitis; meningitis; encephalitis; uveitis; Colitis; gastritis; glomerulonephritis; allergic constitutions such as eczema and asthma and other conditions involving T cell infiltration and chronic inflammatory reactions; atherosclerosis; leukocyte adhesion deficiency; rheumatoid arthritis (RA ); Systemic lupus erythematosus (SLE); diabetes (eg type 1 diabetes or insulin-dependent diabetes); multiple sclerosis; Reynaud's syndrome; autoimmune thyroiditis; allergic encephalomyelitis; Sjogren's syndrome; juvenile-onset diabetes; and mediated by cytokines and T lymphocytes, usually found in tuberculosis, sarcomatosis, polymyositis, granuloma, and vasculitis Immune reactions related to acute and delayed allergic reactions; Wegener's disease; Pernicious anemia (Addison's disease); Diseases involving leukocyte exudation; Inflammatory disorders of the central nervous system (CNS) ; Multiple organ injury syndrome; Hemolytic anemia (including but not limited to cryoglobulinemia or Qom's-positive anemia); Myasthenia gravis; antigen-antibody complex-mediated disease; anti-glomerular basement membrane disease; anti Phospholipid syndrome; allergic neuritis; Graves' disease; Lambert-Eaton myasthenic syndrome; bullous pemphigoid; pemphigus; autoimmune multiple endocrine lesions Leukoplakia; Reiter's disease; Zombie syndrome; Bechet disease; giant cell arteritis; immune complex nephritis; IgA nephropathy; IgM polyneuropathy; immune thrombocytopenic purpura (ITP) or autoimmune thrombocytopenia and autoimmune hemolytic disease; Hashimoto's thyroiditis; autoimmune hepatitis; autoimmune hemophilia; autoimmune lymphoproliferative syndrome (ALPS); autoimmune uveal membrane Retinitis; Guillain-Barre syndrome; Goodpasture's syndrome; mixed nodules Associated tissue disease; infertility related to autoimmunity; polyarteritis nodosa; alopecia areata; idiopathic mucinous edema; graft-versus-host disease; muscular dystrophy (Duchen's type, Becker type, muscle tone type, (Leg strap type, scapular arm type, congenital type, ophthalmopharyngeal type, distal type, Ai-De type) and cancer cells that control CXCR5, such as pancreatic cancer, colon cancer, bladder cancer, T cell leukemia and B The proliferation of cell leukemia. The method of claim 21, wherein the disease is SLE or rheumatoid arthritis. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 or claim 17 in the manufacture of a medicament for the treatment of immune diseases, disorders or conditions. A method for detecting CXCR5 in a sample, tissue or cell using the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, which comprises contacting the sample, tissue or cell with the antibody and detecting the antibody . A method for reducing the biological activity of CXCR5 in an individual in need thereof, the method comprising administering a therapeutically effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 or claim 17, or as claimed The pharmaceutical composition of 18. The method of claim 25, wherein the antibody mediates the depletion of at least one cell expressing CXCR5 selected from the group consisting of Tfh cells in the spleen, B cells in peripheral blood, and Tfh-like cells in peripheral blood. A method of inhibiting a humoral immune response in an individual in need thereof, the method comprising administering a therapeutically effective amount of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 5 or claim 17, or as claimed The pharmaceutical composition of 18. The method of claim 27, wherein the antibody mediates depletion of at least one cell expressing CXCR5 selected from the group consisting of Tfh cells in the spleen, B cells in peripheral blood, and Tfh-like cells in peripheral blood.